dmem f12  (Thermo Fisher)


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    Thermo Fisher dmem f12
    Growth and proliferation of esophagogastric junction cells in vitro . (A) Cells obtained by EI in sub-culture. (A-a) Cell morphology of each group after sub-culture to the third generation. Magnification, x200; scale bar, 200 µm. (A-b) Cell proliferation curves in SMCM (third generation; red curve) and <t>DMEM/F12</t> containing 10% newborn bovine serum (10%-F12; second generation; black curve). n=7. Experiments were perfomed in duplicate. A typical ‘S’ curve was observed in SMCM; in 10%-F12, cell proliferation was largely stopped. (B) Representative cell morphology of EC muscle cells. (B-a) Typical structure of ‘hills and valleys’ for primary (two images above on 10th day after adherence) and sub-cultured (two images below, second generation) cells obtained by EI. Scale bar, 200 µm. Cells cultured in vitro grew in a uniform direction as ‘hills and valleys’, as determined by in topographical mapping. (B-b) An illustration of cell morphology. Magnification, x400; scale bar, 200 µm. Cells were spindle- or long-spindle-shaped, but not uniform; some were rod- or besom-like. Pseudopods of cells differed. (B-c) As the number of passages increased, spindle cells became larger and deformed. Magnification, x200; scale bar, 200 µm. Sizes and morphologies of the fifth (middle) and eighth (right) generation cells were compared with primary spindle cells (left), after 36 h of adherence. EI, enzyme-injected; SMCM, smooth muscle cell medium; Clasp, clasp fiber; Sling, sling fiber; EC, esophageal circular; EL, esophageal longitudinal; GC-S, gastric circular muscle near sling in gastric bottom; GC-C, gastric circular muscle near clasp in lesser gastric curvature; OD, optical density; 0.5-EI-4, 0.5 mg/ml collagenase II solution injected into tissues at 4˚C; 1-EI-4, 1 mg/ml collagenase II solution injected into the tissues at 4˚C; T, Trypsin.
    Dmem F12, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction"

    Article Title: Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2020.8560

    Growth and proliferation of esophagogastric junction cells in vitro . (A) Cells obtained by EI in sub-culture. (A-a) Cell morphology of each group after sub-culture to the third generation. Magnification, x200; scale bar, 200 µm. (A-b) Cell proliferation curves in SMCM (third generation; red curve) and DMEM/F12 containing 10% newborn bovine serum (10%-F12; second generation; black curve). n=7. Experiments were perfomed in duplicate. A typical ‘S’ curve was observed in SMCM; in 10%-F12, cell proliferation was largely stopped. (B) Representative cell morphology of EC muscle cells. (B-a) Typical structure of ‘hills and valleys’ for primary (two images above on 10th day after adherence) and sub-cultured (two images below, second generation) cells obtained by EI. Scale bar, 200 µm. Cells cultured in vitro grew in a uniform direction as ‘hills and valleys’, as determined by in topographical mapping. (B-b) An illustration of cell morphology. Magnification, x400; scale bar, 200 µm. Cells were spindle- or long-spindle-shaped, but not uniform; some were rod- or besom-like. Pseudopods of cells differed. (B-c) As the number of passages increased, spindle cells became larger and deformed. Magnification, x200; scale bar, 200 µm. Sizes and morphologies of the fifth (middle) and eighth (right) generation cells were compared with primary spindle cells (left), after 36 h of adherence. EI, enzyme-injected; SMCM, smooth muscle cell medium; Clasp, clasp fiber; Sling, sling fiber; EC, esophageal circular; EL, esophageal longitudinal; GC-S, gastric circular muscle near sling in gastric bottom; GC-C, gastric circular muscle near clasp in lesser gastric curvature; OD, optical density; 0.5-EI-4, 0.5 mg/ml collagenase II solution injected into tissues at 4˚C; 1-EI-4, 1 mg/ml collagenase II solution injected into the tissues at 4˚C; T, Trypsin.
    Figure Legend Snippet: Growth and proliferation of esophagogastric junction cells in vitro . (A) Cells obtained by EI in sub-culture. (A-a) Cell morphology of each group after sub-culture to the third generation. Magnification, x200; scale bar, 200 µm. (A-b) Cell proliferation curves in SMCM (third generation; red curve) and DMEM/F12 containing 10% newborn bovine serum (10%-F12; second generation; black curve). n=7. Experiments were perfomed in duplicate. A typical ‘S’ curve was observed in SMCM; in 10%-F12, cell proliferation was largely stopped. (B) Representative cell morphology of EC muscle cells. (B-a) Typical structure of ‘hills and valleys’ for primary (two images above on 10th day after adherence) and sub-cultured (two images below, second generation) cells obtained by EI. Scale bar, 200 µm. Cells cultured in vitro grew in a uniform direction as ‘hills and valleys’, as determined by in topographical mapping. (B-b) An illustration of cell morphology. Magnification, x400; scale bar, 200 µm. Cells were spindle- or long-spindle-shaped, but not uniform; some were rod- or besom-like. Pseudopods of cells differed. (B-c) As the number of passages increased, spindle cells became larger and deformed. Magnification, x200; scale bar, 200 µm. Sizes and morphologies of the fifth (middle) and eighth (right) generation cells were compared with primary spindle cells (left), after 36 h of adherence. EI, enzyme-injected; SMCM, smooth muscle cell medium; Clasp, clasp fiber; Sling, sling fiber; EC, esophageal circular; EL, esophageal longitudinal; GC-S, gastric circular muscle near sling in gastric bottom; GC-C, gastric circular muscle near clasp in lesser gastric curvature; OD, optical density; 0.5-EI-4, 0.5 mg/ml collagenase II solution injected into tissues at 4˚C; 1-EI-4, 1 mg/ml collagenase II solution injected into the tissues at 4˚C; T, Trypsin.

    Techniques Used: In Vitro, Cell Culture, Injection

    2) Product Images from "Effects of ICI 182,780, an ERα and ERβ antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells"

    Article Title: Effects of ICI 182,780, an ERα and ERβ antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells

    Journal: Einstein

    doi: 10.31744/einstein_journal/2020AO4560

    Efeitos de ICI, G-1 e rapamicina em células SKBr3. Viabilidade celular por MTT de células SKBr3 tratadas com ICI (A), G-1 (B) e rapamicina (C) por 24, 48 e 72 horas, em meio DMEM/F12, sem soro fetal bovino. Os dados representam a média de um experimento em triplicata. Valor de p
    Figure Legend Snippet: Efeitos de ICI, G-1 e rapamicina em células SKBr3. Viabilidade celular por MTT de células SKBr3 tratadas com ICI (A), G-1 (B) e rapamicina (C) por 24, 48 e 72 horas, em meio DMEM/F12, sem soro fetal bovino. Os dados representam a média de um experimento em triplicata. Valor de p

    Techniques Used: MTT Assay

    Effects of ICI, G-1 and rapamycin in SKBr3 cells. Cell viability by MTT in SKBr3 cells treated with ICI (A), G-1 (B) and rapamycin (C) for 24, 48 and 72 hours in FBS-free DMEM/F12. The data represent the mean of an experiment in triplicate. p
    Figure Legend Snippet: Effects of ICI, G-1 and rapamycin in SKBr3 cells. Cell viability by MTT in SKBr3 cells treated with ICI (A), G-1 (B) and rapamycin (C) for 24, 48 and 72 hours in FBS-free DMEM/F12. The data represent the mean of an experiment in triplicate. p

    Techniques Used: MTT Assay

    Autophagy induced by rapamycin based on GFP-LC3 puncta in MCF-7 cells. ICI and G-1 did not induce autophagy within the time observed (4 hours). In A, images obtained by fluorescence microscopy of GFP-LC3-expressing MCF-7 cells stimulated with rapamycin (1µM), ICI (100nM) and G-1 (100nM). Cells were kept in DMEM/F12 (CTR) and visualized every 30 minutes, over the course of 4 hours (40X magnification). In B, the histogram represents the quantification of cells with increased GFP-LC3 puncta, within 4 hours in MCF-7 cells. The mean±standard error of the mean was calculated from five independent control experiments. The upper limit was obtained (mean + two times the standard error of the mean) and established as cutoff which, in this case, was equal to an increase of nine puncta per cell. For each experiment, we calculated the percentage of cells with an increase greater than this limit. One-way analysis of variance, followed by Bonferroni’s test. Mean values obtained from at least three independent experiments. The ICI, G-1 and rapamycin groups were compared with the Control Group * p
    Figure Legend Snippet: Autophagy induced by rapamycin based on GFP-LC3 puncta in MCF-7 cells. ICI and G-1 did not induce autophagy within the time observed (4 hours). In A, images obtained by fluorescence microscopy of GFP-LC3-expressing MCF-7 cells stimulated with rapamycin (1µM), ICI (100nM) and G-1 (100nM). Cells were kept in DMEM/F12 (CTR) and visualized every 30 minutes, over the course of 4 hours (40X magnification). In B, the histogram represents the quantification of cells with increased GFP-LC3 puncta, within 4 hours in MCF-7 cells. The mean±standard error of the mean was calculated from five independent control experiments. The upper limit was obtained (mean + two times the standard error of the mean) and established as cutoff which, in this case, was equal to an increase of nine puncta per cell. For each experiment, we calculated the percentage of cells with an increase greater than this limit. One-way analysis of variance, followed by Bonferroni’s test. Mean values obtained from at least three independent experiments. The ICI, G-1 and rapamycin groups were compared with the Control Group * p

    Techniques Used: Fluorescence, Microscopy, Expressing

    Expression of estrogen receptors in MCF-7 and SKBr3 cell lines and cell viability by MTT in MCF-7 cells. In A, expression of estrogen receptors alpha and G-protein-coupled receptors (GPER) in MCF-7 and SKBr3 cells. Representative autoradiograms show that estrogen receptor alpha is differentially expressed in the two cell lines, whereas the G-protein-coupled receptor (GPER) is expressed at similar levels. In B, C and D, cell viability by MTT in MCF-7 cells treated with ICI (B), G-1 (C) and rapamycin (D) for 24, 48 and 72 hours in FBS-free DMEM/F12. The data represent the mean±standard error of the mean of four independent experiments (24 hours and 48 hours) and three experiments in triplicate (72 hours). One-way analysis of variance (ICI and G-1) with multiple comparisons by Tukey’s test. Unpaired t test (rapamycin versus CTR). P
    Figure Legend Snippet: Expression of estrogen receptors in MCF-7 and SKBr3 cell lines and cell viability by MTT in MCF-7 cells. In A, expression of estrogen receptors alpha and G-protein-coupled receptors (GPER) in MCF-7 and SKBr3 cells. Representative autoradiograms show that estrogen receptor alpha is differentially expressed in the two cell lines, whereas the G-protein-coupled receptor (GPER) is expressed at similar levels. In B, C and D, cell viability by MTT in MCF-7 cells treated with ICI (B), G-1 (C) and rapamycin (D) for 24, 48 and 72 hours in FBS-free DMEM/F12. The data represent the mean±standard error of the mean of four independent experiments (24 hours and 48 hours) and three experiments in triplicate (72 hours). One-way analysis of variance (ICI and G-1) with multiple comparisons by Tukey’s test. Unpaired t test (rapamycin versus CTR). P

    Techniques Used: Expressing, MTT Assay

    Expressão de receptores de estrógeno nas linhagens MCF-7 e SKBr3 e viabilidade celular por MTT em células MCF-7. Em A, expressão de receptores de estrógeno alfa e receptores acoplados a proteínas-G em células MCF-7 e SKBr3. Os autorradiogramas representativos mostram a expressão diferencial de receptores de estrógeno alfa nas duas linhagens, enquanto o receptor acoplado a proteínas-G se mantém em níveis similares. Em B, C e D, viabilidade celular por MTT de células MCF-7 tratadas com ICI (B), G-1 (C) e rapamicina (D) por 24 horas, 48 horas e 72 horas em meio DMEM/F12 sem soro fetal bovino. Os dados representam a média±erro médio padrão de quatro experimentos independentes (24 horas e 48 horas) e três experimentos (72 horas) feitos em triplicata. Análise de variância uma via (ICI e G-1) com múltiplas comparações por teste Tukey. Teste t não pareado (rapamicina versus CTR). Valor de p
    Figure Legend Snippet: Expressão de receptores de estrógeno nas linhagens MCF-7 e SKBr3 e viabilidade celular por MTT em células MCF-7. Em A, expressão de receptores de estrógeno alfa e receptores acoplados a proteínas-G em células MCF-7 e SKBr3. Os autorradiogramas representativos mostram a expressão diferencial de receptores de estrógeno alfa nas duas linhagens, enquanto o receptor acoplado a proteínas-G se mantém em níveis similares. Em B, C e D, viabilidade celular por MTT de células MCF-7 tratadas com ICI (B), G-1 (C) e rapamicina (D) por 24 horas, 48 horas e 72 horas em meio DMEM/F12 sem soro fetal bovino. Os dados representam a média±erro médio padrão de quatro experimentos independentes (24 horas e 48 horas) e três experimentos (72 horas) feitos em triplicata. Análise de variância uma via (ICI e G-1) com múltiplas comparações por teste Tukey. Teste t não pareado (rapamicina versus CTR). Valor de p

    Techniques Used: MTT Assay

    Indução de autofagia induzida por rapamicina por meio da pontuação de GFP-LC3 em células MCF-7. Observa-se que ICI e G-1 não induziram autofagia no tempo observado (4 horas). Em A, imagens obtidas por microscopia de fluorescência de células MCF-7 expressando GFP-LC3 e estimuladas com rapamicina (1µM), ICI (100nM) e G-1 (100nM). Células foram mantidas em DMEM/F12 (CTR) e visualizadas a cada 30 minutos, durante 4 horas. (objetiva de 40x). Em B, histograma representando a quantificação de células que apresentaram aumento na pontuação de GFP-LC3 por 4 horas em células MCF-7. A média±erro médio padrão foi calculada a partir de cinco experimentos controles independentes. O limite superior foi obtido (média + duas vezes o erro médio padrão) e estabelecido como limite de corte, que, nesse caso, foi igual a um aumento de nove pontuações por célula. Para cada experimento, foi calculada a percentagem de células com aumento superior a esse limite. Análise de variância de uma via, seguida do teste de Bonferroni. Valores médios obtidos de pelo menos três experimentos independentes. Os grupos ICI, G-1 e rapamicina foram comparados ao Grupo Controle *p
    Figure Legend Snippet: Indução de autofagia induzida por rapamicina por meio da pontuação de GFP-LC3 em células MCF-7. Observa-se que ICI e G-1 não induziram autofagia no tempo observado (4 horas). Em A, imagens obtidas por microscopia de fluorescência de células MCF-7 expressando GFP-LC3 e estimuladas com rapamicina (1µM), ICI (100nM) e G-1 (100nM). Células foram mantidas em DMEM/F12 (CTR) e visualizadas a cada 30 minutos, durante 4 horas. (objetiva de 40x). Em B, histograma representando a quantificação de células que apresentaram aumento na pontuação de GFP-LC3 por 4 horas em células MCF-7. A média±erro médio padrão foi calculada a partir de cinco experimentos controles independentes. O limite superior foi obtido (média + duas vezes o erro médio padrão) e estabelecido como limite de corte, que, nesse caso, foi igual a um aumento de nove pontuações por célula. Para cada experimento, foi calculada a percentagem de células com aumento superior a esse limite. Análise de variância de uma via, seguida do teste de Bonferroni. Valores médios obtidos de pelo menos três experimentos independentes. Os grupos ICI, G-1 e rapamicina foram comparados ao Grupo Controle *p

    Techniques Used:

    3) Product Images from "Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium"

    Article Title: Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2012-0163

    Sheets of RPE progenitors are efficiently generated and begin to pigment by day 14 of differentiation. (A): Bright field and immunofluorescence images of a sheet of RPE at day 14. The arrow points to pigmented cells. Scale bars = 500 μm. (B): RPE gene expression following differentiation in insulin-like growth factor 1, Noggin, DKK1, basic fibroblast growth factor, nicotinamide, Activin A, SU5402, and vasoactive intestinal peptide compared with differentiation in basal medium alone. mRNA levels were quantified by quantitative polymerase chain reaction and normalized to the no factor condition (differentiated in B27/N2 DMEM/F12 medium). The error bars represent the standard error of the mean. *, p ≤ .1; **, p ≤ .05. (C): Immunofluorescence images of RPE sheets at day 14. Scale bar = 100 μm. (D): Quantification of Pmel17 and Oct4 immunoreactivity by flow cytometry after 14 days differentiation of H9, UCSF4, and IMR90, compared with undifferentiated H9. The error bars represent the standard error of the mean. **, p ≤ .05. (E): Representative flow cytometry histograms for Pmel17 and Oct4 at day 14 (H9-RPE, H9 no factor differentiation), undifferentiated H9 cells, MeWo cells (positive control for Pmel17), and Hs27 cells (negative control for both Pmel17 and Oct4). Abbreviations: Ctrl, control; RPE, retinal pigmented epithelium.
    Figure Legend Snippet: Sheets of RPE progenitors are efficiently generated and begin to pigment by day 14 of differentiation. (A): Bright field and immunofluorescence images of a sheet of RPE at day 14. The arrow points to pigmented cells. Scale bars = 500 μm. (B): RPE gene expression following differentiation in insulin-like growth factor 1, Noggin, DKK1, basic fibroblast growth factor, nicotinamide, Activin A, SU5402, and vasoactive intestinal peptide compared with differentiation in basal medium alone. mRNA levels were quantified by quantitative polymerase chain reaction and normalized to the no factor condition (differentiated in B27/N2 DMEM/F12 medium). The error bars represent the standard error of the mean. *, p ≤ .1; **, p ≤ .05. (C): Immunofluorescence images of RPE sheets at day 14. Scale bar = 100 μm. (D): Quantification of Pmel17 and Oct4 immunoreactivity by flow cytometry after 14 days differentiation of H9, UCSF4, and IMR90, compared with undifferentiated H9. The error bars represent the standard error of the mean. **, p ≤ .05. (E): Representative flow cytometry histograms for Pmel17 and Oct4 at day 14 (H9-RPE, H9 no factor differentiation), undifferentiated H9 cells, MeWo cells (positive control for Pmel17), and Hs27 cells (negative control for both Pmel17 and Oct4). Abbreviations: Ctrl, control; RPE, retinal pigmented epithelium.

    Techniques Used: Generated, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Positive Control, Negative Control

    4) Product Images from "Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons"

    Article Title: Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13764-8

    TSP1 immunodepletion or TSP1 knockout abolished ACM-exerted effects, which was rescued by the supplementation of exogenous TSP1 protein. ( a ) TSP1 antibodies were used for western blotting, which showed clear TSP1 bands in TSP1, mock, and ACM groups, a weak band in TSP1 immunodepleted ACM group (TSP1-ID-ACM), and no bands in empty, DMEM/F12, or TSP1 knockout ACM (TSP1-KO-ACM) groups. The image was cropped and inverted from a digital image captured by UVP imaging system (original images with ladder in the supplemental Fig. 1 ). ( b ) Immunofluorescence shows TUJ1-expressing connections between EGFP-ScNs (asterisks) and wild type CN neurons (arrowheads). A number of SV2-expressing puncta were observed along connections in the mock group (co-cultures supplied with the wild type ACM that has been immunodepleted by mouse IgG). The number of SV2-expressing puncta (arrows) along connections was reduced in the TSP1-ID-ACM group (co-cultures supplied with TSP1-ID-ACM), whereas it was increased when exogenous TSP1 protein (10 nM) was supplemented (the rescue group). ( b1 ) The quantitative study indicates that the relative number and area of SV2 puncta decreased in the TSP1-ID-ACM group, whereas they significantly increased in the rescue group (mean ± standard error shown in the figure; **indicates P
    Figure Legend Snippet: TSP1 immunodepletion or TSP1 knockout abolished ACM-exerted effects, which was rescued by the supplementation of exogenous TSP1 protein. ( a ) TSP1 antibodies were used for western blotting, which showed clear TSP1 bands in TSP1, mock, and ACM groups, a weak band in TSP1 immunodepleted ACM group (TSP1-ID-ACM), and no bands in empty, DMEM/F12, or TSP1 knockout ACM (TSP1-KO-ACM) groups. The image was cropped and inverted from a digital image captured by UVP imaging system (original images with ladder in the supplemental Fig. 1 ). ( b ) Immunofluorescence shows TUJ1-expressing connections between EGFP-ScNs (asterisks) and wild type CN neurons (arrowheads). A number of SV2-expressing puncta were observed along connections in the mock group (co-cultures supplied with the wild type ACM that has been immunodepleted by mouse IgG). The number of SV2-expressing puncta (arrows) along connections was reduced in the TSP1-ID-ACM group (co-cultures supplied with TSP1-ID-ACM), whereas it was increased when exogenous TSP1 protein (10 nM) was supplemented (the rescue group). ( b1 ) The quantitative study indicates that the relative number and area of SV2 puncta decreased in the TSP1-ID-ACM group, whereas they significantly increased in the rescue group (mean ± standard error shown in the figure; **indicates P

    Techniques Used: Knock-Out, Western Blot, Imaging, Immunofluorescence, Expressing

    5) Product Images from "Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes"

    Article Title: Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes

    Journal: Stem Cells International

    doi: 10.1155/2018/7609284

    Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P
    Figure Legend Snippet: Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P

    Techniques Used: Cell Culture

    qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value
    Figure Legend Snippet: qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Two Tailed Test

    6) Product Images from "KDM5B Promotes Drug Resistance by Regulating Melanoma Propagating Cell Subpopulations"

    Article Title: KDM5B Promotes Drug Resistance by Regulating Melanoma Propagating Cell Subpopulations

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-18-0395

    BRAFi induced reversible upregulation of KDM5B. (A) BRAFi induced upregulation of Kdm5b mRNA level in both CD34 + and CD34 − cells sorted after three days of BRAFi treatment. (*, p≤0.05; ***, p≤0.001; ****, p≤0.0001). (B) KDM5B protein level was upregulated in established clone of CD34 + YUMM1.7 cells (left panel) and unsorted YUMM3.3 cells (right panel) by treatment with 1.5 μM BRAFi for three days. H3K4me3 levels decreased correspondingly in these BRAFi-treated cells. (C) YUMM1.7R cells cultured in 2 μM BRAFi media expressed high levels of KDM5B and had lower level of H3K4me3 than YUMM1.7 cells. (D) Withdrawing 1.5 μM BRAFi from YUMM1.7 and YUMM3.3 after three days of treatment and culturing them in regular DMEM/F12 media with vehicle control DMSO for additional six days restored the Kdm5b level to the baseline level (**, p≤0.01; ***, p≤0.001; n.s., not significant). (E) Replacement of 2 μM BRAFi media with regular DMEM/F12 media with vehicle control DMSO in YUMM1.7R for six days reduced Kdm5b level (****, p≤0.0001; n.s., not significant).
    Figure Legend Snippet: BRAFi induced reversible upregulation of KDM5B. (A) BRAFi induced upregulation of Kdm5b mRNA level in both CD34 + and CD34 − cells sorted after three days of BRAFi treatment. (*, p≤0.05; ***, p≤0.001; ****, p≤0.0001). (B) KDM5B protein level was upregulated in established clone of CD34 + YUMM1.7 cells (left panel) and unsorted YUMM3.3 cells (right panel) by treatment with 1.5 μM BRAFi for three days. H3K4me3 levels decreased correspondingly in these BRAFi-treated cells. (C) YUMM1.7R cells cultured in 2 μM BRAFi media expressed high levels of KDM5B and had lower level of H3K4me3 than YUMM1.7 cells. (D) Withdrawing 1.5 μM BRAFi from YUMM1.7 and YUMM3.3 after three days of treatment and culturing them in regular DMEM/F12 media with vehicle control DMSO for additional six days restored the Kdm5b level to the baseline level (**, p≤0.01; ***, p≤0.001; n.s., not significant). (E) Replacement of 2 μM BRAFi media with regular DMEM/F12 media with vehicle control DMSO in YUMM1.7R for six days reduced Kdm5b level (****, p≤0.0001; n.s., not significant).

    Techniques Used: Cell Culture

    7) Product Images from "Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors"

    Article Title: Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123193

    Autonomous hepatic differentiation of hiHSCs in Essential 6 medium. Self-renewing hiHSCs (clone AFB1-1) differentiate into hepatocyte-like cells in defined Essential 6 medium that consists simply of insulin, transferrin, selenium, and L-ascorbic acid in DMEM/F12 medium. ( A–G ) Gene expression was analyzed by quantitative RT-PCR at day 12. Relative expression is shown in the histogram. The expression is normalized to 1 in the self-renewing hiHSCs (mTeSR1/MEF) and compared to that of hepatocyte-like cells (E6). Gene symbols are shown for ( A ) serum hepatic proteins, ( B ) cytochrome P450 enzymes, ( C ) conjugating enzymes and transporters, ( D ) hepatic transcription factors, ( E ) endodermal transcription factors, ( F ) ESC transcription factors, and ( G ) urea cycle-related enzymes. Data are presented as mean+SEM and represent a minimum of three independent samples with at least two technical duplicates. ( H ) Urea production was measured by urea nitrogen detection kits on the supernatants of cells at day 12. E6 fresh medium was also measured as a negative control. Data are presented as mean+SEM and represent a minimum of three independent samples. ( I ) Immunofluorescent staining of hepatocyte-like cells for FABP1, ALB, CK8, and ASGPR1. Scale bar represents 100 μm. ( J ) Release of ALB was measured by ELISA at three time points. Data are presented as mean±SEM and represent a minimum of three independent samples with at least two technical duplicates. See also Table 1 . ( K ) Phase contrast micrographs showing the morphology of differentiating cells. Scale bar represents 100 μm. ( L ) At day 12, hepatocyte-like cells are shown to store glycogen by Periodic Acid—Schiff (PAS) staining. Otherwise, the cells were stained to uptake indocyanine green (ICG) for 5 hr and BODIPY FL–labeled low density lipoprotein (LDL) for 48 hr. Scale bar represents 100 μm.
    Figure Legend Snippet: Autonomous hepatic differentiation of hiHSCs in Essential 6 medium. Self-renewing hiHSCs (clone AFB1-1) differentiate into hepatocyte-like cells in defined Essential 6 medium that consists simply of insulin, transferrin, selenium, and L-ascorbic acid in DMEM/F12 medium. ( A–G ) Gene expression was analyzed by quantitative RT-PCR at day 12. Relative expression is shown in the histogram. The expression is normalized to 1 in the self-renewing hiHSCs (mTeSR1/MEF) and compared to that of hepatocyte-like cells (E6). Gene symbols are shown for ( A ) serum hepatic proteins, ( B ) cytochrome P450 enzymes, ( C ) conjugating enzymes and transporters, ( D ) hepatic transcription factors, ( E ) endodermal transcription factors, ( F ) ESC transcription factors, and ( G ) urea cycle-related enzymes. Data are presented as mean+SEM and represent a minimum of three independent samples with at least two technical duplicates. ( H ) Urea production was measured by urea nitrogen detection kits on the supernatants of cells at day 12. E6 fresh medium was also measured as a negative control. Data are presented as mean+SEM and represent a minimum of three independent samples. ( I ) Immunofluorescent staining of hepatocyte-like cells for FABP1, ALB, CK8, and ASGPR1. Scale bar represents 100 μm. ( J ) Release of ALB was measured by ELISA at three time points. Data are presented as mean±SEM and represent a minimum of three independent samples with at least two technical duplicates. See also Table 1 . ( K ) Phase contrast micrographs showing the morphology of differentiating cells. Scale bar represents 100 μm. ( L ) At day 12, hepatocyte-like cells are shown to store glycogen by Periodic Acid—Schiff (PAS) staining. Otherwise, the cells were stained to uptake indocyanine green (ICG) for 5 hr and BODIPY FL–labeled low density lipoprotein (LDL) for 48 hr. Scale bar represents 100 μm.

    Techniques Used: Expressing, Quantitative RT-PCR, Negative Control, Staining, Enzyme-linked Immunosorbent Assay, Labeling

    8) Product Images from "Paracrine effects of human amniotic epithelial cells protect against chemotherapy-induced ovarian damage"

    Article Title: Paracrine effects of human amniotic epithelial cells protect against chemotherapy-induced ovarian damage

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0721-0

    Effects of hAECs and hAEC-CM on follicle development and gene expression in the injured ovaries. A Representative photomicrographs of H E-stained ovarian sections came from left and right ovary obtained from the same mouse in different treatment groups. Pictures b, d, f, h, j, l, n, p, r and t were magnifications of the square in photos a, c, e, g, i, k, m, o, q and s, respectively. B Real-time PCR was used to detect the expression of follicle growth-related genes in the injured ovarian tissues at 1 month after hAECs or hAEC-CM treatment, respectively. Data is represented means ± SEM. Sham group, n = 10; Cy + PBS group, n = 10; Cy + hAECs group, n = 10; Cy + DMEM/F12 group, n = 6; Cy + hAEC-CM group, n = 10. * P
    Figure Legend Snippet: Effects of hAECs and hAEC-CM on follicle development and gene expression in the injured ovaries. A Representative photomicrographs of H E-stained ovarian sections came from left and right ovary obtained from the same mouse in different treatment groups. Pictures b, d, f, h, j, l, n, p, r and t were magnifications of the square in photos a, c, e, g, i, k, m, o, q and s, respectively. B Real-time PCR was used to detect the expression of follicle growth-related genes in the injured ovarian tissues at 1 month after hAECs or hAEC-CM treatment, respectively. Data is represented means ± SEM. Sham group, n = 10; Cy + PBS group, n = 10; Cy + hAECs group, n = 10; Cy + DMEM/F12 group, n = 6; Cy + hAEC-CM group, n = 10. * P

    Techniques Used: Expressing, Staining, Real-time Polymerase Chain Reaction

    9) Product Images from "The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response"

    Article Title: The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23169287

    ( A ) Changes in the actin cytoskeleton architecture of hUC-MSCs cultured on selected surfaces with immobilized rhBMP-2. Cells were cultured in a medium supplemented with 2% FBS for 24 h and were then immunostained for vinculin and counterstained with TRITC-phalloidin (F-actin) and DAPI (nuclei). The scale bar represents 25 μm. ( B ) Cell area after 24 h of seeding measured for 30 cells for each condition, shown as the fold-change vs. appropriate pure polymeric systems (controls). ( C ) Expression of osteogenic markers in hUC-MSCs cultured on selected surfaces for seven days. Quantitative real-time PCR analysis of selected osteogenic markers (ALPL–alkaline phosphatase; COL10A1–collagen type 10; OCN–osteocalcin) in hUC-MSCs cultured in DMEM/F12, 2% FBS. Gene expression was normalized to the expression of a housekeeping gene–GADPH, shown as the fold-change compared to gene expression in hUC-MSC cultured on the clean substrate–glass coverslips (blue line). p values less than 0.05 ( p
    Figure Legend Snippet: ( A ) Changes in the actin cytoskeleton architecture of hUC-MSCs cultured on selected surfaces with immobilized rhBMP-2. Cells were cultured in a medium supplemented with 2% FBS for 24 h and were then immunostained for vinculin and counterstained with TRITC-phalloidin (F-actin) and DAPI (nuclei). The scale bar represents 25 μm. ( B ) Cell area after 24 h of seeding measured for 30 cells for each condition, shown as the fold-change vs. appropriate pure polymeric systems (controls). ( C ) Expression of osteogenic markers in hUC-MSCs cultured on selected surfaces for seven days. Quantitative real-time PCR analysis of selected osteogenic markers (ALPL–alkaline phosphatase; COL10A1–collagen type 10; OCN–osteocalcin) in hUC-MSCs cultured in DMEM/F12, 2% FBS. Gene expression was normalized to the expression of a housekeeping gene–GADPH, shown as the fold-change compared to gene expression in hUC-MSC cultured on the clean substrate–glass coverslips (blue line). p values less than 0.05 ( p

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    ( A ) Cell area 24 h after seeding. ( B ) DNA concentration of hUC-MSC proliferation evaluated after 24, 48, 72 h, and 7 days of cell-culture on a clean substrate, (DR/CS) 6 and (DR/CS) 6 DR. ( C ) Expression of osteogenic markers in hUC-MSCs cultured on a clean substrate, (DR/CS) 6 , and (DR/CS) 6 DR for 7 days in vitro. Quantitative real-time PCR analysis of selected osteogenic markers (ALPL–alkaline phosphatase; COL10A1–collagen type 10; OCN–osteocalcin) in hUC-MSCs cultured in DMEM/F12, 2% FBS). Gene expression was normalized to the expression of a housekeeping gene–GADPH, shown as the fold-change compared to gene expression in hUC-MSCs cultured on the clean substrate. p values less than 0.05 ( p
    Figure Legend Snippet: ( A ) Cell area 24 h after seeding. ( B ) DNA concentration of hUC-MSC proliferation evaluated after 24, 48, 72 h, and 7 days of cell-culture on a clean substrate, (DR/CS) 6 and (DR/CS) 6 DR. ( C ) Expression of osteogenic markers in hUC-MSCs cultured on a clean substrate, (DR/CS) 6 , and (DR/CS) 6 DR for 7 days in vitro. Quantitative real-time PCR analysis of selected osteogenic markers (ALPL–alkaline phosphatase; COL10A1–collagen type 10; OCN–osteocalcin) in hUC-MSCs cultured in DMEM/F12, 2% FBS). Gene expression was normalized to the expression of a housekeeping gene–GADPH, shown as the fold-change compared to gene expression in hUC-MSCs cultured on the clean substrate. p values less than 0.05 ( p

    Techniques Used: Concentration Assay, Cell Culture, Expressing, In Vitro, Real-time Polymerase Chain Reaction

    10) Product Images from "Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro"

    Article Title: Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038243

    The growth and differentiation of NSCs with different media at 3 days in vitro . (A) Differentiation of NSCs co-cultured with protoplasmic astrocytes. (B) Differentiation of NSCs in NB+N2 medium. (C) Differentiation of NSCs in co-culture+BDNF antibody medium. (D) Differentiation of NSCs in DMEM/F12+10% FBS medium. The scale bar = 150 µm.
    Figure Legend Snippet: The growth and differentiation of NSCs with different media at 3 days in vitro . (A) Differentiation of NSCs co-cultured with protoplasmic astrocytes. (B) Differentiation of NSCs in NB+N2 medium. (C) Differentiation of NSCs in co-culture+BDNF antibody medium. (D) Differentiation of NSCs in DMEM/F12+10% FBS medium. The scale bar = 150 µm.

    Techniques Used: In Vitro, Cell Culture, Co-Culture Assay

    Quantification of differentiated cells cultured in various media at 7 days in vitro . Protoplasmic astrocytes grown in co-culture medium (A), NB+B27 medium (B), co-culture medium+BDNF antibody (C), and DMEM/F12+10% FBS medium (D). Scale bar = 75 µm. (E) Quantification of differentiated neurons in the various media conditions. β-tubulin III staining (green) indicates neurons; GFAP staining (red) indicates astrocytes. The nuclei were counterstained with DAPI (blue). *p
    Figure Legend Snippet: Quantification of differentiated cells cultured in various media at 7 days in vitro . Protoplasmic astrocytes grown in co-culture medium (A), NB+B27 medium (B), co-culture medium+BDNF antibody (C), and DMEM/F12+10% FBS medium (D). Scale bar = 75 µm. (E) Quantification of differentiated neurons in the various media conditions. β-tubulin III staining (green) indicates neurons; GFAP staining (red) indicates astrocytes. The nuclei were counterstained with DAPI (blue). *p

    Techniques Used: Cell Culture, In Vitro, Co-Culture Assay, Staining

    11) Product Images from "Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes"

    Article Title: Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes

    Journal: Stem Cells International

    doi: 10.1155/2018/7609284

    Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P
    Figure Legend Snippet: Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P

    Techniques Used: Cell Culture

    qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value
    Figure Legend Snippet: qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Two Tailed Test

    12) Product Images from "Curcumin-incorporated 3D bioprinting gelatin methacryloyl hydrogel reduces reactive oxygen species-induced adipose-derived stem cell apoptosis and improves implanting survival in diabetic wounds"

    Article Title: Curcumin-incorporated 3D bioprinting gelatin methacryloyl hydrogel reduces reactive oxygen species-induced adipose-derived stem cell apoptosis and improves implanting survival in diabetic wounds

    Journal: Burns & Trauma

    doi: 10.1093/burnst/tkac001

    Structure of GelMA and Cur-GelMA scaffolds. ( a ) Scanning electron microscopy images of 10% GelMA and 10% Cur-GelMA. ( b, c ) Quantification of the pore size and porosity of GelMA and Cur-GelMA scaffolds. ( d ) Scanning electron microscopy images of Cur in GelMA scaffold showing smooth surface morphologies. The mean diameter of particles was 0.99 ± 0.12 μm. ( e ) Quantification of the swelling ratio of GelMA and Cur-GelMA scaffolds. ( f ) Representative images of ADSCs on 10% GelMA surfaces. ( g–i ) Typical atomic force microscopy curve and Young’s modulus of GelMA. ( j ) Printability test of 10% GelMA and 10% Cur-GelMA hydrogels. ( k ) Cur degrades in DMEM/F12 with 10% FBS. GelMA Gelatin methacryloyl, Cur curcumin, ADSC adipose-derived stem cell
    Figure Legend Snippet: Structure of GelMA and Cur-GelMA scaffolds. ( a ) Scanning electron microscopy images of 10% GelMA and 10% Cur-GelMA. ( b, c ) Quantification of the pore size and porosity of GelMA and Cur-GelMA scaffolds. ( d ) Scanning electron microscopy images of Cur in GelMA scaffold showing smooth surface morphologies. The mean diameter of particles was 0.99 ± 0.12 μm. ( e ) Quantification of the swelling ratio of GelMA and Cur-GelMA scaffolds. ( f ) Representative images of ADSCs on 10% GelMA surfaces. ( g–i ) Typical atomic force microscopy curve and Young’s modulus of GelMA. ( j ) Printability test of 10% GelMA and 10% Cur-GelMA hydrogels. ( k ) Cur degrades in DMEM/F12 with 10% FBS. GelMA Gelatin methacryloyl, Cur curcumin, ADSC adipose-derived stem cell

    Techniques Used: Electron Microscopy, Microscopy, Derivative Assay

    13) Product Images from "AN OPTIMIZED SMALL MOLECULE INHIBITOR COCKTAIL SUPPORTS LONG-TERM MAINTENANCE OF HUMAN EMBRYONIC STEM CELLS"

    Article Title: AN OPTIMIZED SMALL MOLECULE INHIBITOR COCKTAIL SUPPORTS LONG-TERM MAINTENANCE OF HUMAN EMBRYONIC STEM CELLS

    Journal: Nature communications

    doi: 10.1038/ncomms1165

    OCT4-GFP expression-based optimization. ( a ) Combination 40300 was tested in various media and on different extracellular matrix proteins. KSR, DMEM/F12 supplemented with 20% knockout serum replacement; N2B27, DMEM/F12 supplemented with N2 and B27 supplements; Minimal, DMEM/F12 supplemented with transferrin, albumin and insulin. Results were presented as the percentage of OCT4-GFP + cells within the total number of viable cells. ( b ) After several OCT4-GFP-based iterations, top combinations (black arrows) were selected based on the total number of OCT4-GFP positive cells. Data is normalized to the gold standard (GS, red column) and can be directly compared to the starting combination 40300 (green column); 00000, no small molecules added. ( c ) Short term passaging of H1-OCT4-GFP and HSF1-OCT4-GFP hES cells in GS and 4 selected combinations. All combinations, except for 43300 with H1-OCT4-GFP cells, failed to support high expressions of OCT4-GFP over three continuous passages. Combination 43300, despite supporting a higher percentage of OCT4-GFP + cells, could not deliver a sufficient number of viable cells after 3 passages.
    Figure Legend Snippet: OCT4-GFP expression-based optimization. ( a ) Combination 40300 was tested in various media and on different extracellular matrix proteins. KSR, DMEM/F12 supplemented with 20% knockout serum replacement; N2B27, DMEM/F12 supplemented with N2 and B27 supplements; Minimal, DMEM/F12 supplemented with transferrin, albumin and insulin. Results were presented as the percentage of OCT4-GFP + cells within the total number of viable cells. ( b ) After several OCT4-GFP-based iterations, top combinations (black arrows) were selected based on the total number of OCT4-GFP positive cells. Data is normalized to the gold standard (GS, red column) and can be directly compared to the starting combination 40300 (green column); 00000, no small molecules added. ( c ) Short term passaging of H1-OCT4-GFP and HSF1-OCT4-GFP hES cells in GS and 4 selected combinations. All combinations, except for 43300 with H1-OCT4-GFP cells, failed to support high expressions of OCT4-GFP over three continuous passages. Combination 43300, despite supporting a higher percentage of OCT4-GFP + cells, could not deliver a sufficient number of viable cells after 3 passages.

    Techniques Used: Expressing, Knock-Out, Passaging

    14) Product Images from "Loss of Alpha3(IV) Collagen Expression Associated with Corneal Keratocyte Activation"

    Article Title: Loss of Alpha3(IV) Collagen Expression Associated with Corneal Keratocyte Activation

    Journal: Investigative ophthalmology & visual science

    doi: 10.1167/iovs.06-0635

    Growth of rabbit corneal keratocytes under different culture conditions. Keratocytes isolated from rabbit corneas were plated in DMEM/F12 without FBS. After 24 hours, culture medium was replaced with DMEM/F12 without FBS or supplemented with 10 µg/mL
    Figure Legend Snippet: Growth of rabbit corneal keratocytes under different culture conditions. Keratocytes isolated from rabbit corneas were plated in DMEM/F12 without FBS. After 24 hours, culture medium was replaced with DMEM/F12 without FBS or supplemented with 10 µg/mL

    Techniques Used: Isolation

    Phase-contrast images ( A–E ) and actin filament staining patterns ( F–J ) of rabbit corneal stromal cells in culture. Keratocytes isolated from rabbit corneas were plated in serum-free DMEM/F12 and allowed to attach for 24 hours. The medium
    Figure Legend Snippet: Phase-contrast images ( A–E ) and actin filament staining patterns ( F–J ) of rabbit corneal stromal cells in culture. Keratocytes isolated from rabbit corneas were plated in serum-free DMEM/F12 and allowed to attach for 24 hours. The medium

    Techniques Used: Staining, Isolation

    Western blot analyses of the α3(IV) collagen in cell extracts of rabbit corneal stromal cells cultured in serum-free DMEM/F12(SF) or in DMEM/F12 supplemented with insulin, 10% fetal bovine serum (FBS), bFGF (20 ng/mL)/HS (5 µg/mL), or
    Figure Legend Snippet: Western blot analyses of the α3(IV) collagen in cell extracts of rabbit corneal stromal cells cultured in serum-free DMEM/F12(SF) or in DMEM/F12 supplemented with insulin, 10% fetal bovine serum (FBS), bFGF (20 ng/mL)/HS (5 µg/mL), or

    Techniques Used: Western Blot, Cell Culture

    Immunocytochemical analyses of corneal stromal cells in culture. Stromal cells isolated from rabbit corneas were cultured in DMEM/F12 without FBS ( A–C ), with 10 µg/mL insulin ( D–F ), with 10% FBS ( G–I ), with bFGF (20 ng/mL)/HS
    Figure Legend Snippet: Immunocytochemical analyses of corneal stromal cells in culture. Stromal cells isolated from rabbit corneas were cultured in DMEM/F12 without FBS ( A–C ), with 10 µg/mL insulin ( D–F ), with 10% FBS ( G–I ), with bFGF (20 ng/mL)/HS

    Techniques Used: Isolation, Cell Culture

    Western blot analyses of KSPGs in the culture supernatants of rabbit corneal stromal cells. Top : Anti-KS reactive bands in the culture supernatants of rabbit corneal stromal cells cultured in DMEM/F12 without serum (SF) or with 10 µg/mL insulin,
    Figure Legend Snippet: Western blot analyses of KSPGs in the culture supernatants of rabbit corneal stromal cells. Top : Anti-KS reactive bands in the culture supernatants of rabbit corneal stromal cells cultured in DMEM/F12 without serum (SF) or with 10 µg/mL insulin,

    Techniques Used: Western Blot, Cell Culture

    15) Product Images from "A Novel Role of IL13Rα2 in the Pathogenesis of Proliferative Vitreoretinopathy"

    Article Title: A Novel Role of IL13Rα2 in the Pathogenesis of Proliferative Vitreoretinopathy

    Journal: Frontiers in Medicine

    doi: 10.3389/fmed.2022.831436

    Vitreous induced the protein expression of IL13Rα2. Western blot analysis of the vitreous-induced protein expression of IL13RA2 in ARPE-19 cells. One of three representative experiments is shown. Bar graphs showed the Western blot band intensity, and the mean ± SD of three independent experiments is shown; *** denotes 0.001, using paired t test. (A) The protein expression of IL13RA2 in ARPE-19 cells. (B) Histogram of protein expression gray value. The mean ± SD of three independent experiments is shown; *** denotes 0.001, using paired t test. Control: ARPE-19 cells treated with DMEM/F12 only. Vitreous: ARPE-19 cells treated with vitreous diluted in DMEM/F12.
    Figure Legend Snippet: Vitreous induced the protein expression of IL13Rα2. Western blot analysis of the vitreous-induced protein expression of IL13RA2 in ARPE-19 cells. One of three representative experiments is shown. Bar graphs showed the Western blot band intensity, and the mean ± SD of three independent experiments is shown; *** denotes 0.001, using paired t test. (A) The protein expression of IL13RA2 in ARPE-19 cells. (B) Histogram of protein expression gray value. The mean ± SD of three independent experiments is shown; *** denotes 0.001, using paired t test. Control: ARPE-19 cells treated with DMEM/F12 only. Vitreous: ARPE-19 cells treated with vitreous diluted in DMEM/F12.

    Techniques Used: Expressing, Western Blot

    Blocking IL13Rα2 prevents vitreous-induced contraction of RPE cells. After the mixtures of ARPE-19 cells with collagen I solution formed a collagen gel, they were treated with DMEM/F12 + an antibody or vitreous + an antibody for 48 h. C, control antibody; N-A: neutralizing antibody against IL13Rα2; V + C, vitreous + control antibody; V + N-A, vitreous + neutralizing antibody against IL13Rα2. *** denotes 0.001, using paired t test.
    Figure Legend Snippet: Blocking IL13Rα2 prevents vitreous-induced contraction of RPE cells. After the mixtures of ARPE-19 cells with collagen I solution formed a collagen gel, they were treated with DMEM/F12 + an antibody or vitreous + an antibody for 48 h. C, control antibody; N-A: neutralizing antibody against IL13Rα2; V + C, vitreous + control antibody; V + N-A, vitreous + neutralizing antibody against IL13Rα2. *** denotes 0.001, using paired t test.

    Techniques Used: Blocking Assay

    16) Product Images from "Alginate encapsulated multipotent adult progenitor cells promote corneal stromal cell activation via release of soluble factors"

    Article Title: Alginate encapsulated multipotent adult progenitor cells promote corneal stromal cell activation via release of soluble factors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202118

    A schematic representation of the encapsulation and storage process of alginate with or without MAPC. (A) The initial gelling of the alginate prepared by transferring the mixed alginate with or without MAPC into the CaCl 2 masked filter paper and left for two minutes before allocating the gel into 4 ml CaCl 2 for final gelling. (B) The formed encapsulations are either stored in a cryovials or in an air tight chamber for 72 hours at (4 and 15°C). (C) Following corneal stromal cell scratch-wound induction, the gel is situated on the upper compartment of the transwell to avoid direct contact between the gel and the corneal stromal cells monolayer. MAPC, Multipotent adult progenitor cells; SFM, serum-free DMEM:F12 medium; XFM, xenobiotic-free MAPC medium.
    Figure Legend Snippet: A schematic representation of the encapsulation and storage process of alginate with or without MAPC. (A) The initial gelling of the alginate prepared by transferring the mixed alginate with or without MAPC into the CaCl 2 masked filter paper and left for two minutes before allocating the gel into 4 ml CaCl 2 for final gelling. (B) The formed encapsulations are either stored in a cryovials or in an air tight chamber for 72 hours at (4 and 15°C). (C) Following corneal stromal cell scratch-wound induction, the gel is situated on the upper compartment of the transwell to avoid direct contact between the gel and the corneal stromal cells monolayer. MAPC, Multipotent adult progenitor cells; SFM, serum-free DMEM:F12 medium; XFM, xenobiotic-free MAPC medium.

    Techniques Used: Transferring

    17) Product Images from "WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro"

    Article Title: WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro

    Journal: Cells

    doi: 10.3390/cells11152431

    Neurosphere stemness induction in low propagating ANPGs from a C57Bl/6 mouse . ( A ) C57Bl/6 ANPG were plated at 10,000/well of a 96-well plate and treated with WNT agonist (CHIR99021; 3 µM) and/or TGFβ Smad antagonist (dual SMAD inhibitors; LDN193189 0.5 µM, SB431542 10 µM) aiming at replicating phoenix ANPG pattern of gene expression and subsequent stemness phenotype. ( B ) Growth of ANPG neurospheres was followed twice a week over 38 days and the bar graph shows the average growth/day. Phoenix cells are used as positive control. C57Bl/6: ANPGs were cultured in DMEM:F12 + N2 and B27 supplements without growth factors. +GF: DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF (previously described conditions). GF + DS:DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF + LDN193189 0.5 µM + SB431542 10 µM. GF + CHIR: DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF + CHIR99021 3 µM. +GF + CHIR + DS: DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF + LDN193189 0.5 µM + SB431542 10 µM + CHIR99021 3 µM. Phoenix +GF: phoenix ANPG cultured in standard conditions (DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF). The data represent the average ± SEM of three independent experiments. ( C ) At every passage, following dissociation with Accutase, ANPGs were counted with a fast-read counting chamber. Graph showing the number of cells/passage up to passage 5. Whereas C57Bl/6 ANPGs cultured in standard conditions were not able to expand (see bottom line representing + GF; and middle line representing +GF + CHIR), stemness induced ANPGs exhibited exponential growth (green line (ANPGs + GF + CHIR + DS)). The data represent the average ± SEM of three independent experiments. * p
    Figure Legend Snippet: Neurosphere stemness induction in low propagating ANPGs from a C57Bl/6 mouse . ( A ) C57Bl/6 ANPG were plated at 10,000/well of a 96-well plate and treated with WNT agonist (CHIR99021; 3 µM) and/or TGFβ Smad antagonist (dual SMAD inhibitors; LDN193189 0.5 µM, SB431542 10 µM) aiming at replicating phoenix ANPG pattern of gene expression and subsequent stemness phenotype. ( B ) Growth of ANPG neurospheres was followed twice a week over 38 days and the bar graph shows the average growth/day. Phoenix cells are used as positive control. C57Bl/6: ANPGs were cultured in DMEM:F12 + N2 and B27 supplements without growth factors. +GF: DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF (previously described conditions). GF + DS:DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF + LDN193189 0.5 µM + SB431542 10 µM. GF + CHIR: DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF + CHIR99021 3 µM. +GF + CHIR + DS: DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF + LDN193189 0.5 µM + SB431542 10 µM + CHIR99021 3 µM. Phoenix +GF: phoenix ANPG cultured in standard conditions (DMEM:F12 + N2 and B27 + IGF + EFG + HS + FGF). The data represent the average ± SEM of three independent experiments. ( C ) At every passage, following dissociation with Accutase, ANPGs were counted with a fast-read counting chamber. Graph showing the number of cells/passage up to passage 5. Whereas C57Bl/6 ANPGs cultured in standard conditions were not able to expand (see bottom line representing + GF; and middle line representing +GF + CHIR), stemness induced ANPGs exhibited exponential growth (green line (ANPGs + GF + CHIR + DS)). The data represent the average ± SEM of three independent experiments. * p

    Techniques Used: Expressing, Positive Control, Cell Culture

    18) Product Images from "Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy"

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2019/9290728

    Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P
    Figure Legend Snippet: Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Techniques Used: Transmission Electron Microscopy, Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    19) Product Images from "Angiotensin-( - - ) and angiotensin II induce the transdifferentiation of human endometrial epithelial cells in vitro"

    Article Title: Angiotensin-( - - ) and angiotensin II induce the transdifferentiation of human endometrial epithelial cells in vitro

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2014.2128

    EEC proliferation assessed using an MTT assay. EECs were incubated with different intervention factors for the indicated durations (48–72 h). The groups were as follows: Control, EECs incubated with serum-free DMEM/F12; AngII, EECs incubated with 10 −6 mol/l AngII and serum-free DMEM/F12; Ang-( 1 – 7 ), EECs incubated with 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12; AngII+Ang-( 1 – 7 ), EECs incubated with 10 −6 mol/l AngII and 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12. Values are presented as the mean ± standard deviation (n=6). * P
    Figure Legend Snippet: EEC proliferation assessed using an MTT assay. EECs were incubated with different intervention factors for the indicated durations (48–72 h). The groups were as follows: Control, EECs incubated with serum-free DMEM/F12; AngII, EECs incubated with 10 −6 mol/l AngII and serum-free DMEM/F12; Ang-( 1 – 7 ), EECs incubated with 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12; AngII+Ang-( 1 – 7 ), EECs incubated with 10 −6 mol/l AngII and 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12. Values are presented as the mean ± standard deviation (n=6). * P

    Techniques Used: MTT Assay, Incubation, Standard Deviation

    Protein expression of α-SMA and E-cadherin in EECs. EECs were incubated with different intervention factors for 72 h. (A) Immunocytochemistry staining showed brown-yellow granules in the cytoplasm of the EECs (magnification, ×400). (B) Levels of α-SMA and E-cadherin in the EECs, quantified by calculating the average integral A values. The groups were as follows: Control, EECs incubated with serum-free DMEM/F12; AngII, EECs incubated with 10 −6 mol/l Ang II and serum-free DMEM/F12; Ang-( 1 – 7 ), EECs incubated with 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12; Ang II+Ang-( 1 – 7 ), EECs incubated with 10 −6 mol/l Ang II and 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12. Values are presented as the mean ± standard deviation (n=6). * P
    Figure Legend Snippet: Protein expression of α-SMA and E-cadherin in EECs. EECs were incubated with different intervention factors for 72 h. (A) Immunocytochemistry staining showed brown-yellow granules in the cytoplasm of the EECs (magnification, ×400). (B) Levels of α-SMA and E-cadherin in the EECs, quantified by calculating the average integral A values. The groups were as follows: Control, EECs incubated with serum-free DMEM/F12; AngII, EECs incubated with 10 −6 mol/l Ang II and serum-free DMEM/F12; Ang-( 1 – 7 ), EECs incubated with 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12; Ang II+Ang-( 1 – 7 ), EECs incubated with 10 −6 mol/l Ang II and 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12. Values are presented as the mean ± standard deviation (n=6). * P

    Techniques Used: Expressing, Incubation, Immunocytochemistry, Staining, Standard Deviation

    Ang-( 1 – 7 ) inhibits Ang II-induced EEC transdifferentiation. EECs were incubated with different intervention factors for 72 h. (A) The protein expression of α-SMA and E-cadherin was analyzed using western blot analysis. (B) The protein expression of α-SMA and E-cadherin were quantified using densitometry. (C) The levels of Col I and FN protein in the EEC culture supernatant detected using ELISA. (D) Col I, FN, α-SMA and E-cadherin mRNA expression analyzed using quantitative polymerase chain reaction. The groups were as follows: Control, EECs incubated with serum-free DMEM/F12; Ang II, EECs incubated with 10-6 mol/l Ang II and serum-free DMEM/F12; Ang-( 1 – 7 ), EECs incubated with 10-5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12; Ang II+Ang-( 1 – 7 ), EECs incubated with 10-6 mol/l Ang II and 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12. Values are presented as the mean ± standard deviation (n=6). * P
    Figure Legend Snippet: Ang-( 1 – 7 ) inhibits Ang II-induced EEC transdifferentiation. EECs were incubated with different intervention factors for 72 h. (A) The protein expression of α-SMA and E-cadherin was analyzed using western blot analysis. (B) The protein expression of α-SMA and E-cadherin were quantified using densitometry. (C) The levels of Col I and FN protein in the EEC culture supernatant detected using ELISA. (D) Col I, FN, α-SMA and E-cadherin mRNA expression analyzed using quantitative polymerase chain reaction. The groups were as follows: Control, EECs incubated with serum-free DMEM/F12; Ang II, EECs incubated with 10-6 mol/l Ang II and serum-free DMEM/F12; Ang-( 1 – 7 ), EECs incubated with 10-5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12; Ang II+Ang-( 1 – 7 ), EECs incubated with 10-6 mol/l Ang II and 10 −5 mol/l Ang-( 1 – 7 ) and serum-free DMEM/F12. Values are presented as the mean ± standard deviation (n=6). * P

    Techniques Used: Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    20) Product Images from "Mesenchymal Stem Cells Ameliorate Th1-Induced Pre-Eclampsia-Like Symptoms in Mice via the Suppression of TNF-? Expression"

    Article Title: Mesenchymal Stem Cells Ameliorate Th1-Induced Pre-Eclampsia-Like Symptoms in Mice via the Suppression of TNF-? Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088036

    Isolation and characterization of MSCs derived from human decidua. (A): Schematic description of the way to isolate MSCs. (B): Morphology of MSCs isolate after 4 day, 6 day and 8 day (20X). (C) Cell growth curve of MSCs. UCMSCs of passage 2 were plated in 24-well plates in DMEM/F12 supplemented with 10% FBS at a density of 5×10 3 cells/well. The cells were detached with 0.25% trypsin-EDTA every 2 days and cell counting until 14 day. Bars show the mean. n = 3. (D) Osteogenic differentiation was assayed by the alizarin red staining. No mineralized matrix formation was found in UCMSCs maintained in regular growth medium. Osteogenic differentiation was evidenced by the formation of mineralized matrix in UCMSCs after osteogeneic induction. (10X). Scale bars, 100 µm. (E) Adipogenic differentiation was detected by oil red O staining. No lipid vacuoles were found in in UCMSCs maintained in the regular medium. Adipogenic differentiation was evidenced by the formation of lipid vacuoles by oil-red O staining in UCMSCs after adipogenic induction. (10X) Scale bars, 100 µm. All data are representative of three independent experiments.
    Figure Legend Snippet: Isolation and characterization of MSCs derived from human decidua. (A): Schematic description of the way to isolate MSCs. (B): Morphology of MSCs isolate after 4 day, 6 day and 8 day (20X). (C) Cell growth curve of MSCs. UCMSCs of passage 2 were plated in 24-well plates in DMEM/F12 supplemented with 10% FBS at a density of 5×10 3 cells/well. The cells were detached with 0.25% trypsin-EDTA every 2 days and cell counting until 14 day. Bars show the mean. n = 3. (D) Osteogenic differentiation was assayed by the alizarin red staining. No mineralized matrix formation was found in UCMSCs maintained in regular growth medium. Osteogenic differentiation was evidenced by the formation of mineralized matrix in UCMSCs after osteogeneic induction. (10X). Scale bars, 100 µm. (E) Adipogenic differentiation was detected by oil red O staining. No lipid vacuoles were found in in UCMSCs maintained in the regular medium. Adipogenic differentiation was evidenced by the formation of lipid vacuoles by oil-red O staining in UCMSCs after adipogenic induction. (10X) Scale bars, 100 µm. All data are representative of three independent experiments.

    Techniques Used: Isolation, Derivative Assay, Cell Counting, Staining

    21) Product Images from "Olfactory Horizontal Basal Cells Demonstrate a Conserved Multipotent Progenitor Phenotype"

    Article Title: Olfactory Horizontal Basal Cells Demonstrate a Conserved Multipotent Progenitor Phenotype

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0330-04.2004

    Cells from the ICAM-1-rich fraction of OE form highly adhesive clonal colonies and produce differentiated neurons and glia in vitro. A , At P5, the HBC layer contains ICAM-1 (green)/β 4 integrin (red)-rich cells on a laminin-rich (blue) basal lamina. B , ICAM-1 HBCs are dividing in a direction perpendicular to the basal lamina (double arrow), where one daughter contains β 4 integrin (red) and the other does not. C , Some regions contain HBC with ICAM-1 and β 4 expression primarily restricted to the basal surface. D - F , The ICAM-1+ (green)/cytokeratin+ (red) is a small percentage of the total population (DAPI; blue) but represents the majority of PCNA/Ki67-positive cells ( E ) that are clearly undergoing mitosis ( * on HBC of origin) in a plane perpendicular to the basal lamina ( F ). An ICAM-1+-rich/NCAM-negative cell fraction prepared from the neonatal mouse OE was subsequently dissociated into single cells and plated at clonal density on a collagen matrix in DMEM/F12 with 10% fetal calf serum. G , Single cells ( * marks the same cell position followed over time in vitro ) of this cell fraction expanded rapidly over 7 DIV to form small, tightly adhesive colonies. H , Colony expansion continued from 7 to 14 DIV to produce colonies containing 25-40,000 cells (medium-large colony shown here). C , At 14-28 DIV, the larger colonies (colony marked with * in top left-hand corner) produced multiple differentiated progeny ( I-K ) (bipolar and multipolar), which usually migrated away from the edge the colony. J begins at the edge of the original colony boundary. In some instances, differentiated progeny arose from the center of the colony and migrated along the more adhesive colonies beneath. L, M , Cells of both glial ( L ) (S100β+) and neuronal ( M ) (type III β neuron-specific tubulin (TuJ1+) phenotypes were represented in migratory cells. Scale bars: B, C, F , 10 μm; G - M , 50 μm.
    Figure Legend Snippet: Cells from the ICAM-1-rich fraction of OE form highly adhesive clonal colonies and produce differentiated neurons and glia in vitro. A , At P5, the HBC layer contains ICAM-1 (green)/β 4 integrin (red)-rich cells on a laminin-rich (blue) basal lamina. B , ICAM-1 HBCs are dividing in a direction perpendicular to the basal lamina (double arrow), where one daughter contains β 4 integrin (red) and the other does not. C , Some regions contain HBC with ICAM-1 and β 4 expression primarily restricted to the basal surface. D - F , The ICAM-1+ (green)/cytokeratin+ (red) is a small percentage of the total population (DAPI; blue) but represents the majority of PCNA/Ki67-positive cells ( E ) that are clearly undergoing mitosis ( * on HBC of origin) in a plane perpendicular to the basal lamina ( F ). An ICAM-1+-rich/NCAM-negative cell fraction prepared from the neonatal mouse OE was subsequently dissociated into single cells and plated at clonal density on a collagen matrix in DMEM/F12 with 10% fetal calf serum. G , Single cells ( * marks the same cell position followed over time in vitro ) of this cell fraction expanded rapidly over 7 DIV to form small, tightly adhesive colonies. H , Colony expansion continued from 7 to 14 DIV to produce colonies containing 25-40,000 cells (medium-large colony shown here). C , At 14-28 DIV, the larger colonies (colony marked with * in top left-hand corner) produced multiple differentiated progeny ( I-K ) (bipolar and multipolar), which usually migrated away from the edge the colony. J begins at the edge of the original colony boundary. In some instances, differentiated progeny arose from the center of the colony and migrated along the more adhesive colonies beneath. L, M , Cells of both glial ( L ) (S100β+) and neuronal ( M ) (type III β neuron-specific tubulin (TuJ1+) phenotypes were represented in migratory cells. Scale bars: B, C, F , 10 μm; G - M , 50 μm.

    Techniques Used: In Vitro, Expressing, Produced

    22) Product Images from "Atypical Cadherin FAT3 Is a Novel Mediator for Morphological Changes of Microglia"

    Article Title: Atypical Cadherin FAT3 Is a Novel Mediator for Morphological Changes of Microglia

    Journal: eNeuro

    doi: 10.1523/ENEURO.0056-20.2020

    Hypoxanthine promotes FAT3 expression in microglia. A , The chart summarizes the additional components of DMEM/F12 against DMEM. They are categorized into three large groups, non-essential amino acids (NEAAs), metallic ions (metals), and the others. B , BV2 cells were incubated with the indicated components for 24 h. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least three independent experiments. C , BV2 cells were incubated with 15 μM hypoxanthine (HYP), 0.5 μM vitamin B12 (B12), 15 nM biotin, 0.15 μM linoleic acid (Lino), or 0.5 μM lipoic acid (Lipo) for 24 h. The FAT3 mRNAs were measured by quantitative RT-PCR analysis and were normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least four independent experiments. D , BV2 cells were incubated with either DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 24 h. Immunostaining showing the FAT3 localization in the presence or absence of 15 μM hypoxanthine. Scale bar: 50 μm. E , Phase contrast images showing BV2 morphology. Scale bar: 50 μm. F , Quantification of data from E . The score indicates a ratio of maximum to minimum Feret diameter; n > 68 cells; 5 fields. The elongated cell is defined as the score over 2. G , The graph shows the relative percentage of the elongated cells in each field; n = 5 fields; mean ± SD; p value was calculated by one-way ANOVA. The data were reproduced in at least four independent experiments. H , The primary microglia were incubated with either DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 24 h. Immunostaining showing the FAT3 expression in the presence or absence of 15 μM hypoxanthine. Scale bar: 50 μm. I , The primary microglia were incubated with either DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 24 h. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in two independent experiments.
    Figure Legend Snippet: Hypoxanthine promotes FAT3 expression in microglia. A , The chart summarizes the additional components of DMEM/F12 against DMEM. They are categorized into three large groups, non-essential amino acids (NEAAs), metallic ions (metals), and the others. B , BV2 cells were incubated with the indicated components for 24 h. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least three independent experiments. C , BV2 cells were incubated with 15 μM hypoxanthine (HYP), 0.5 μM vitamin B12 (B12), 15 nM biotin, 0.15 μM linoleic acid (Lino), or 0.5 μM lipoic acid (Lipo) for 24 h. The FAT3 mRNAs were measured by quantitative RT-PCR analysis and were normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least four independent experiments. D , BV2 cells were incubated with either DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 24 h. Immunostaining showing the FAT3 localization in the presence or absence of 15 μM hypoxanthine. Scale bar: 50 μm. E , Phase contrast images showing BV2 morphology. Scale bar: 50 μm. F , Quantification of data from E . The score indicates a ratio of maximum to minimum Feret diameter; n > 68 cells; 5 fields. The elongated cell is defined as the score over 2. G , The graph shows the relative percentage of the elongated cells in each field; n = 5 fields; mean ± SD; p value was calculated by one-way ANOVA. The data were reproduced in at least four independent experiments. H , The primary microglia were incubated with either DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 24 h. Immunostaining showing the FAT3 expression in the presence or absence of 15 μM hypoxanthine. Scale bar: 50 μm. I , The primary microglia were incubated with either DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 24 h. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in two independent experiments.

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Immunostaining

    FAT3 regulates BV2 morphology. A , BV2 cells were incubated with either DMEM as control medium or high-nutrient medium, DMEM/F12 for 24 h. Scale bar: 50 μm. B , Quantification of data from A . The score indicates a ratio of maximum to minimum Feret diameter; n > 78 cells; 5 fields. The elongated cell is defined as the score over 2. C , The graph shows the relative percentage of the elongated cells in each field; n = 5 fields; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least five independent experiments. D , A subset of mRNAs identified in microarray experiments. The graph indicates the top 17 of most upregulated and 11 of most downregulated genes under DMEM/F12 culture condition. E , The graph indicates in silico analysis of microglial FAT3 expression level from P0 to P60 in mice. The values were obtained from the Brain RNA-Seq database ( http://www.brainrnaseq.org/ ). The red line indicates immature microglia (Tmem119 – cells), and the blue line indicates mature microglia (Tmem119 + cells). F , BV2 cells were incubated with either DMEM/F12 or DMEM for 24 h. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least four independent experiments. G , HEK293T cells were transfected with HA-FAT3 CT (4313-4589) and subjected to immunoblot analysis with anti-FAT3, anti-HA, and anti-tubulin antibodies. H , left, BV2 cells were incubated with either DMEM/F12 or DMEM for 24 h. Immunostaining showing the localization of FAT3 cultured in either DMEM or DMEM/F12. Arrow, FAT3 expression in the distal processes and the edge of lamellipodia. Arrowhead, FAT3 in the proximal region. Scale bar: 50 μm. Right, The line profiles of FAT3 staining. The yellow areas indicate the nuclei regions. The orange arrowheads indicate accumulated FAT3 regions. I , BV2 cells were incubated for 24 h after transfection of FAT3 siRNA. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least three independent experiments. J , Immunostaining showing the FAT3 localization in the presence or absence of FAT3 siRNA for 24 h. Scale bar: 50 μm. K , BV2 cells were incubated in the presence or absence of FAT3 siRNA for 24 h. Scale bar: 50 μm. L , Quantification of data from K . The score indicates a ratio of maximum to minimum Feret diameter; n > 32 cells; 10 fields. The elongated cell is defined as the score over 2. M , The graph shows the relative percentage of the elongated cells in each field; n = 10 fields; mean ± SD; p value was calculated by one-way ANOVA. The data were reproduced in at least three independent experiments.
    Figure Legend Snippet: FAT3 regulates BV2 morphology. A , BV2 cells were incubated with either DMEM as control medium or high-nutrient medium, DMEM/F12 for 24 h. Scale bar: 50 μm. B , Quantification of data from A . The score indicates a ratio of maximum to minimum Feret diameter; n > 78 cells; 5 fields. The elongated cell is defined as the score over 2. C , The graph shows the relative percentage of the elongated cells in each field; n = 5 fields; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least five independent experiments. D , A subset of mRNAs identified in microarray experiments. The graph indicates the top 17 of most upregulated and 11 of most downregulated genes under DMEM/F12 culture condition. E , The graph indicates in silico analysis of microglial FAT3 expression level from P0 to P60 in mice. The values were obtained from the Brain RNA-Seq database ( http://www.brainrnaseq.org/ ). The red line indicates immature microglia (Tmem119 – cells), and the blue line indicates mature microglia (Tmem119 + cells). F , BV2 cells were incubated with either DMEM/F12 or DMEM for 24 h. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least four independent experiments. G , HEK293T cells were transfected with HA-FAT3 CT (4313-4589) and subjected to immunoblot analysis with anti-FAT3, anti-HA, and anti-tubulin antibodies. H , left, BV2 cells were incubated with either DMEM/F12 or DMEM for 24 h. Immunostaining showing the localization of FAT3 cultured in either DMEM or DMEM/F12. Arrow, FAT3 expression in the distal processes and the edge of lamellipodia. Arrowhead, FAT3 in the proximal region. Scale bar: 50 μm. Right, The line profiles of FAT3 staining. The yellow areas indicate the nuclei regions. The orange arrowheads indicate accumulated FAT3 regions. I , BV2 cells were incubated for 24 h after transfection of FAT3 siRNA. FAT3 mRNAs were measured by quantitative RT-PCR analysis and normalized to 5S ribosomal RNA; n = 3; mean ± SD. The data were reproduced in at least three independent experiments. J , Immunostaining showing the FAT3 localization in the presence or absence of FAT3 siRNA for 24 h. Scale bar: 50 μm. K , BV2 cells were incubated in the presence or absence of FAT3 siRNA for 24 h. Scale bar: 50 μm. L , Quantification of data from K . The score indicates a ratio of maximum to minimum Feret diameter; n > 32 cells; 10 fields. The elongated cell is defined as the score over 2. M , The graph shows the relative percentage of the elongated cells in each field; n = 10 fields; mean ± SD; p value was calculated by one-way ANOVA. The data were reproduced in at least three independent experiments.

    Techniques Used: Incubation, Microarray, In Silico, Expressing, Mouse Assay, RNA Sequencing Assay, Quantitative RT-PCR, Transfection, Immunostaining, Cell Culture, Staining

    Intracellular hypoxanthine is crucial for BV2 morphologic changes. A , Schematic overview of purinergic metabolisms. R5P; ribose 5-phosphate; PRPP, phosphoribosyl pyrophosphate. B , BV2 cells were incubated in the presence or absence of 15 μM hypoxanthine (HYP) and 10 μM PPADS for 24 h. Scale bar: 50 μm. The data were reproduced in at least three independent experiments. C , Quantification of data from B . The score indicates a ratio of maximum to minimum Feret diameter; n > 57 cells; 5 fields. The elongated cell is defined as the score over 2. D , The graph indicates the relative percentage of elongated BV2 cells in C ; n = 5 fields; mean ± SD; p value was calculated by one-way ANOVA. N.S., not significant. The data were reproduced in at least two independent experiments. E , The graph indicates the intracellular hypoxanthine concentration cultured in DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 3 h; n = 3; mean ± SD; p value was calculated by one-way ANOVA. The data were reproduced in at least two independent experiments . F , The graph indicates the intracellular hypoxanthine concentration in BV2 cells cultured in DMEM in the presence or absence of 10 μM DY for 3 h. Mean ± SD; p value was calculated by Student’s t test. G , BV2 cells were incubated in the presence or absence of 10 μM DY cultured in DMEM for 24 h. Scale bar: 50 μm. H , Quantification of data from G . The score indicates a ratio of maximum to minimum Feret diameter; n > 79 cells; 5 fields. The threshold of the elongated cells was defined by setting over 2. I , The graph indicates the relative percentage of elongated BV2 cells with or without DY in H ; n = 5 fields; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least three independent experiments. J , The graph indicates the intracellular hypoxanthine concentration in the presence or absence of 10 μM ALP cultured in DMEM for 3 h; n = 5; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least two independent experiments. K , BV2 cells were incubated in the presence or absence of 10 μM ALP cultured in DMEM for 24 h. Scale bar: 50 μm. L , Quantification of data from K . The score indicates a ratio of maximum to minimum Feret diameter; n > 79 cells; 5 fields. The elongated cell is defined as the score over 2. M , The graph indicates the relative percentage of elongated BV2 cells in L ; n = 5 fields; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least three independent experiments.
    Figure Legend Snippet: Intracellular hypoxanthine is crucial for BV2 morphologic changes. A , Schematic overview of purinergic metabolisms. R5P; ribose 5-phosphate; PRPP, phosphoribosyl pyrophosphate. B , BV2 cells were incubated in the presence or absence of 15 μM hypoxanthine (HYP) and 10 μM PPADS for 24 h. Scale bar: 50 μm. The data were reproduced in at least three independent experiments. C , Quantification of data from B . The score indicates a ratio of maximum to minimum Feret diameter; n > 57 cells; 5 fields. The elongated cell is defined as the score over 2. D , The graph indicates the relative percentage of elongated BV2 cells in C ; n = 5 fields; mean ± SD; p value was calculated by one-way ANOVA. N.S., not significant. The data were reproduced in at least two independent experiments. E , The graph indicates the intracellular hypoxanthine concentration cultured in DMEM, DMEM/F12, or DMEM with 15 μM hypoxanthine for 3 h; n = 3; mean ± SD; p value was calculated by one-way ANOVA. The data were reproduced in at least two independent experiments . F , The graph indicates the intracellular hypoxanthine concentration in BV2 cells cultured in DMEM in the presence or absence of 10 μM DY for 3 h. Mean ± SD; p value was calculated by Student’s t test. G , BV2 cells were incubated in the presence or absence of 10 μM DY cultured in DMEM for 24 h. Scale bar: 50 μm. H , Quantification of data from G . The score indicates a ratio of maximum to minimum Feret diameter; n > 79 cells; 5 fields. The threshold of the elongated cells was defined by setting over 2. I , The graph indicates the relative percentage of elongated BV2 cells with or without DY in H ; n = 5 fields; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least three independent experiments. J , The graph indicates the intracellular hypoxanthine concentration in the presence or absence of 10 μM ALP cultured in DMEM for 3 h; n = 5; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least two independent experiments. K , BV2 cells were incubated in the presence or absence of 10 μM ALP cultured in DMEM for 24 h. Scale bar: 50 μm. L , Quantification of data from K . The score indicates a ratio of maximum to minimum Feret diameter; n > 79 cells; 5 fields. The elongated cell is defined as the score over 2. M , The graph indicates the relative percentage of elongated BV2 cells in L ; n = 5 fields; mean ± SD; p value was calculated by Student’s t test. The data were reproduced in at least three independent experiments.

    Techniques Used: Incubation, Concentration Assay, Cell Culture

    Hypoxanthine-FAT3 pathway controls the BV2 morphologic dynamics in concert with a humoral factor. A , BV2 cells were imaged at one frame per 15 min over for 12 h with 1.0 μg/μl LPS in the presence or absence of 15 μM hypoxanthine (HYP). Representative images are shown. The blue and red lines indicate the elongated states. Scale bar: 20 μm. B , The graph indicates the ratio of maximum to minimum Feret diameter. C , The graph is summarized by data in B ; n = 4; mean ± SD; p values were calculated by Student’s t test. The data were reproduced in at least three independent experiments. D , Scheme of experiment. BV2 cells were incubated with DMEM/F12 medium for 24 h and were then replaced with either conditioned or fresh media for 24 h. For the conditioned media, other BV2 cells were incubated for 24 h using indicated media. E , BV2 cells were incubated for 6 h after exchanging media and were then subjected to immunocytochemistry. Immunostaining showing the expression level of FAT3. It seems that FAT3 expression is maintained after changing the media. Scale bar: 50 μm. F , Phase contrast images showing BV2 morphology. Scale bar: 50 μm. G , The score indicates a ratio of maximum to minimum Feret diameter; n > 39 cells; 5 fields. The elongated cell is defined as the score over 2. H , The graph indicates the relative percentage of elongated BV2 cells morphology in F ; n = 5 fields; mean ± SD; p values were calculated by one-way ANOVA. N.S., not significant. The data were reproduced in at least two independent experiments.
    Figure Legend Snippet: Hypoxanthine-FAT3 pathway controls the BV2 morphologic dynamics in concert with a humoral factor. A , BV2 cells were imaged at one frame per 15 min over for 12 h with 1.0 μg/μl LPS in the presence or absence of 15 μM hypoxanthine (HYP). Representative images are shown. The blue and red lines indicate the elongated states. Scale bar: 20 μm. B , The graph indicates the ratio of maximum to minimum Feret diameter. C , The graph is summarized by data in B ; n = 4; mean ± SD; p values were calculated by Student’s t test. The data were reproduced in at least three independent experiments. D , Scheme of experiment. BV2 cells were incubated with DMEM/F12 medium for 24 h and were then replaced with either conditioned or fresh media for 24 h. For the conditioned media, other BV2 cells were incubated for 24 h using indicated media. E , BV2 cells were incubated for 6 h after exchanging media and were then subjected to immunocytochemistry. Immunostaining showing the expression level of FAT3. It seems that FAT3 expression is maintained after changing the media. Scale bar: 50 μm. F , Phase contrast images showing BV2 morphology. Scale bar: 50 μm. G , The score indicates a ratio of maximum to minimum Feret diameter; n > 39 cells; 5 fields. The elongated cell is defined as the score over 2. H , The graph indicates the relative percentage of elongated BV2 cells morphology in F ; n = 5 fields; mean ± SD; p values were calculated by one-way ANOVA. N.S., not significant. The data were reproduced in at least two independent experiments.

    Techniques Used: Incubation, Immunocytochemistry, Immunostaining, Expressing

    23) Product Images from "Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors"

    Article Title: Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24959-y

    Stepwise derivation of premyogenic progenitors by CHIR-99021 and LDN-193189. ( A ) Initial four steps of the myogenic differentiation protocol for human iPSCs 19 . Three, 6, 8, and 12 days after starting the induction (▼), cells were collected, and total RNA was extracted for RT-qPCR. D: DMEM/F12, i: ITS, C: CHIR-99021, L: LDN-193189, F: FGF-2, K: KSR, H: HGF, and I: IGF-1. The detailed composition of the medium was described in ref. 19 . ( B ) RT-qPCR analysis of T , TBX6 , PAX3 , and PAX7 expression in 201B7, 454E2, GFPT1 #3, and GFPT1 #8 iPS cell lines at different time points. Data are from three independent experiments. CMS: congenital myasthenic syndrome.
    Figure Legend Snippet: Stepwise derivation of premyogenic progenitors by CHIR-99021 and LDN-193189. ( A ) Initial four steps of the myogenic differentiation protocol for human iPSCs 19 . Three, 6, 8, and 12 days after starting the induction (▼), cells were collected, and total RNA was extracted for RT-qPCR. D: DMEM/F12, i: ITS, C: CHIR-99021, L: LDN-193189, F: FGF-2, K: KSR, H: HGF, and I: IGF-1. The detailed composition of the medium was described in ref. 19 . ( B ) RT-qPCR analysis of T , TBX6 , PAX3 , and PAX7 expression in 201B7, 454E2, GFPT1 #3, and GFPT1 #8 iPS cell lines at different time points. Data are from three independent experiments. CMS: congenital myasthenic syndrome.

    Techniques Used: Quantitative RT-PCR, Expressing

    24) Product Images from "Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes"

    Article Title: Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes

    Journal: Stem Cells International

    doi: 10.1155/2018/7609284

    Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P
    Figure Legend Snippet: Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P

    Techniques Used: Cell Culture

    qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value
    Figure Legend Snippet: qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Two Tailed Test

    25) Product Images from "Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes"

    Article Title: Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes

    Journal: Stem Cells International

    doi: 10.1155/2018/7609284

    Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P
    Figure Legend Snippet: Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P

    Techniques Used: Cell Culture

    qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value
    Figure Legend Snippet: qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Two Tailed Test

    26) Product Images from "Interferon-? Regulates the Proliferation and Differentiation of Mesenchymal Stem Cells via Activation of Indoleamine 2,3 Dioxygenase (IDO)"

    Article Title: Interferon-? Regulates the Proliferation and Differentiation of Mesenchymal Stem Cells via Activation of Indoleamine 2,3 Dioxygenase (IDO)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014698

    IFN-γ inhibits the proliferation of mouse and human MSCs. A and B . Cell growth of human and mouse MSCs showing cumulative population doublings as a function of time in culture. Between P4 and P12, cells were cultured in the continuous presence of IFN-γ (100 IU/ml) and/or IDO inhibitors norharmane, D-1-methyl-tryptophan and L-1-methyl-tryptophan for 80 and 50 days respectively. At every passage, population doubling was calculated by the formula logN/log2 as described by Stenderup [63] where N is the ratio between the number of viable cells reaching confluence and the number of cells initially plated. Medium was changed every three days using α-MEM containing 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, 20% or 10% non-inactivated FBS, for human and mouse cells respectively, specially tested for the ability to sustain the growth of MSCs. C. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without tryptophan and treated for 5 days with increasing concentrations of FBS (0, 0.1, 0.5, 2 or 10%) and IFN-γ (0, 1 or 10 IU/ml) D. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without serum and treated for 5 days with increasing concentrations of tryptophan (0, 0.1, 1, 5, 10 or 44.2 µM) and IFN-γ (0, 1 or 10 IU/ml) E. Number of viable mouse MSCs (passage 14) cultured in serum free DMEM F12 medium in the presence of 10 IU/ml IFN-γ, increasing concentrations of tryptophan (0, 0.1, 1, 5, and 10 µM) and/or IDO inhibitors D-1-methyl-tryptophan and L-1-methyl-tryptophan (100 µM) for 5 days. Mouse MSCs were cultured with 10% FBS as positive controls. F. Expression of full IDO1 mRNA in mouse MSCs (passage 20) as measured by qRT-PCR. Cells were grown in the presence of increasing concentrations of tryptophan (0, 1, 5, 10 and 44 µM) and/or IFN-γ (0, 2, 10 and 100 IU/ml) for 24 hours. Mouse MSCs were cultured with 10% FBS as positive controls. Data are mean ± standard error (SEM). *p
    Figure Legend Snippet: IFN-γ inhibits the proliferation of mouse and human MSCs. A and B . Cell growth of human and mouse MSCs showing cumulative population doublings as a function of time in culture. Between P4 and P12, cells were cultured in the continuous presence of IFN-γ (100 IU/ml) and/or IDO inhibitors norharmane, D-1-methyl-tryptophan and L-1-methyl-tryptophan for 80 and 50 days respectively. At every passage, population doubling was calculated by the formula logN/log2 as described by Stenderup [63] where N is the ratio between the number of viable cells reaching confluence and the number of cells initially plated. Medium was changed every three days using α-MEM containing 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, 20% or 10% non-inactivated FBS, for human and mouse cells respectively, specially tested for the ability to sustain the growth of MSCs. C. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without tryptophan and treated for 5 days with increasing concentrations of FBS (0, 0.1, 0.5, 2 or 10%) and IFN-γ (0, 1 or 10 IU/ml) D. Number of viable cells as measured by Alamar blue in mouse MSC cultures (passage 29) grown in DMEM F12 medium without serum and treated for 5 days with increasing concentrations of tryptophan (0, 0.1, 1, 5, 10 or 44.2 µM) and IFN-γ (0, 1 or 10 IU/ml) E. Number of viable mouse MSCs (passage 14) cultured in serum free DMEM F12 medium in the presence of 10 IU/ml IFN-γ, increasing concentrations of tryptophan (0, 0.1, 1, 5, and 10 µM) and/or IDO inhibitors D-1-methyl-tryptophan and L-1-methyl-tryptophan (100 µM) for 5 days. Mouse MSCs were cultured with 10% FBS as positive controls. F. Expression of full IDO1 mRNA in mouse MSCs (passage 20) as measured by qRT-PCR. Cells were grown in the presence of increasing concentrations of tryptophan (0, 1, 5, 10 and 44 µM) and/or IFN-γ (0, 2, 10 and 100 IU/ml) for 24 hours. Mouse MSCs were cultured with 10% FBS as positive controls. Data are mean ± standard error (SEM). *p

    Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR

    27) Product Images from "The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632"

    Article Title: The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142442

    Culture pTR-E Cells in Different Medium. (A) Adherent cells grown in DMEM supplemented with 10% FBS (M1) at day 7 and day 14. Embryonic bodies like structures formed when bFGF removed from KF medium (M2) at 7 days and 14 days. The scale bar represents 100μm. (B-C) RT-PCR analyses of pTR cells cultured with different medium. CDX2 , ELF5 , HAND1 and TEAD4 were still expressed after differentiation. MASH2 was not expressed in the matured cells cultured in M2. PAG in pIVFTR cells was absent after differentiation in the DMEM/F12/FBS medium for 14 days.
    Figure Legend Snippet: Culture pTR-E Cells in Different Medium. (A) Adherent cells grown in DMEM supplemented with 10% FBS (M1) at day 7 and day 14. Embryonic bodies like structures formed when bFGF removed from KF medium (M2) at 7 days and 14 days. The scale bar represents 100μm. (B-C) RT-PCR analyses of pTR cells cultured with different medium. CDX2 , ELF5 , HAND1 and TEAD4 were still expressed after differentiation. MASH2 was not expressed in the matured cells cultured in M2. PAG in pIVFTR cells was absent after differentiation in the DMEM/F12/FBS medium for 14 days.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture

    28) Product Images from "Inhibition of Histone Deacetylases Antagonized FGF2 and IL-1β Effects on MMP Expression in Human Articular Chondrocytes"

    Article Title: Inhibition of Histone Deacetylases Antagonized FGF2 and IL-1β Effects on MMP Expression in Human Articular Chondrocytes

    Journal: Growth factors (Chur, Switzerland)

    doi: 10.1080/08977190802625179

    Time course of cell growth in human articular chondrocyte cultures. Cells were cultured in 12-well culture plates with 1% FBS DMEM/F12 medium. Triplicate wells received treatment with FGF2 (25 ng/ml) and IL-1β (5 ng/ml) in the presence or absence
    Figure Legend Snippet: Time course of cell growth in human articular chondrocyte cultures. Cells were cultured in 12-well culture plates with 1% FBS DMEM/F12 medium. Triplicate wells received treatment with FGF2 (25 ng/ml) and IL-1β (5 ng/ml) in the presence or absence

    Techniques Used: Cell Culture

    29) Product Images from "Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2)"

    Article Title: Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2)

    Journal: Biomolecules

    doi: 10.3390/biom12020211

    Plasminogen enhances endothelial migration using PAR1 and PAR2. Starved HUVECs, untreated (Ut) or treated with Plg, were allowed to migrate as described in “Material and Methods”. “Alone” denotes HUVECs in diluent of DMEM: F12 + 0.1% BSA. Cells, the Plm inhibitor and PAR antagonists were resuspended in this same medium. ( A ) Plg induced significantly higher endothelial cell migration compared to Ut HUVECs, and this increase was significantly inhibited by an irreversible Plm inhibitor, D-Val-Phe-Lys chloromethylketone dihydrochloride. ( B ) Effects of PAR1 and PAR2 antagonists on HUVEC migration in the presence or absence of Plg. Data are representative of 3 independent experiments, each containing at least triplicates. Values are means ± S.E.
    Figure Legend Snippet: Plasminogen enhances endothelial migration using PAR1 and PAR2. Starved HUVECs, untreated (Ut) or treated with Plg, were allowed to migrate as described in “Material and Methods”. “Alone” denotes HUVECs in diluent of DMEM: F12 + 0.1% BSA. Cells, the Plm inhibitor and PAR antagonists were resuspended in this same medium. ( A ) Plg induced significantly higher endothelial cell migration compared to Ut HUVECs, and this increase was significantly inhibited by an irreversible Plm inhibitor, D-Val-Phe-Lys chloromethylketone dihydrochloride. ( B ) Effects of PAR1 and PAR2 antagonists on HUVEC migration in the presence or absence of Plg. Data are representative of 3 independent experiments, each containing at least triplicates. Values are means ± S.E.

    Techniques Used: Migration

    Plasminogen-induced endothelial cell migration depends on H2B, PAR1 and PAR2. Starved HUVECs, untreated (Ut) or treated with Plg, PAR1 or PAR2 antagonists, mouse Fab or Fab of mAb to H2B, either alone or in combinations, were allowed to migrate in a Boyden chamber. “Alone” denotes HUVECs with mouse IgG or anti-H2B alone. Mouse IgG, H2B IgG, PAR antagonists and cells were resuspended in DMEM: F12 + 0.1% BSA. ( A ) H2B blockade with Fab of anti-H2B significantly inhibited the migration of Plg-treated cells. ( B ) Effect of anti-H2B, PAR1 and PAR2 antagonists on migration facilitated by Plg. Data are representative of 3 independent experiments containing at least triplicates in each. Values are means ± S.E.
    Figure Legend Snippet: Plasminogen-induced endothelial cell migration depends on H2B, PAR1 and PAR2. Starved HUVECs, untreated (Ut) or treated with Plg, PAR1 or PAR2 antagonists, mouse Fab or Fab of mAb to H2B, either alone or in combinations, were allowed to migrate in a Boyden chamber. “Alone” denotes HUVECs with mouse IgG or anti-H2B alone. Mouse IgG, H2B IgG, PAR antagonists and cells were resuspended in DMEM: F12 + 0.1% BSA. ( A ) H2B blockade with Fab of anti-H2B significantly inhibited the migration of Plg-treated cells. ( B ) Effect of anti-H2B, PAR1 and PAR2 antagonists on migration facilitated by Plg. Data are representative of 3 independent experiments containing at least triplicates in each. Values are means ± S.E.

    Techniques Used: Migration

    30) Product Images from "Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development"

    Article Title: Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development

    Journal: Animal Nutrition

    doi: 10.1016/j.aninu.2021.06.001

    Comparison between commercial medium and the assembled medium in culturing chicken crypts. (A) Growth of chicken crypts under different treatments. (B) Number of live organoids obtained with different treatments. a to e Different letters on bars mean a significant difference ( P ≤ 0.05). (C) Mouse crypts cultured for 5 d in the assembled medium but without chicken serum (scale bar, 200 μm). Experiments were performed at least 3 times and a representative one was exhibited. Error bars, SEM ( n = 3 wells). The assembled medium is composed of advanced DMEM/F12 with supplemented with epidermal growth factor, Noggin, R-spondin 1, A8301, SB202190, CHIR99021 and valproic acid. IOGM, a commercial medium named IntestinCult Organoid Growth Medium. ASCV, the mixture of A8301, SB202190, CHIR99021, and valproic acid; CS, chicken serum.
    Figure Legend Snippet: Comparison between commercial medium and the assembled medium in culturing chicken crypts. (A) Growth of chicken crypts under different treatments. (B) Number of live organoids obtained with different treatments. a to e Different letters on bars mean a significant difference ( P ≤ 0.05). (C) Mouse crypts cultured for 5 d in the assembled medium but without chicken serum (scale bar, 200 μm). Experiments were performed at least 3 times and a representative one was exhibited. Error bars, SEM ( n = 3 wells). The assembled medium is composed of advanced DMEM/F12 with supplemented with epidermal growth factor, Noggin, R-spondin 1, A8301, SB202190, CHIR99021 and valproic acid. IOGM, a commercial medium named IntestinCult Organoid Growth Medium. ASCV, the mixture of A8301, SB202190, CHIR99021, and valproic acid; CS, chicken serum.

    Techniques Used: Cell Culture

    31) Product Images from "Bile acid: a potential inducer of colon cancer stem cells"

    Article Title: Bile acid: a potential inducer of colon cancer stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0439-4

    DCA and LCA induce increases in CSC markers. Quantitative real-time PCR showed significantly increased mRNA expression of CD44 ( a ), CD166 ( b ), and ALDHA1 ( c ) in HCoEpiC following incubation with 100 μM DCA or LCA for 72 h. Flow cytometric analysis of HCoEpiC showing an increased proportion of CD44-positive and CD166-positive cells following 12-day incubation in the absence (control) or presence of 100 μM DCA or LCA ( d ). DCA/LCA mediated the increase in spheroid formation by the CD44 + CD166 – CSC phenotype of HCoEpiC ( e ); photomicrographs showing spheroids formed in response to DCA or LCA at the end of the 12-day incubation period. For spheroid formation, CD44 + CD166 – cells were sorted by flow cytometry and 200–300 cells were seeded with B27 containing DMEM/F12 medium in 96-well low-attachment plates; 24 h after seeding, 100 μM DCA or LCA was added to the incubation medium, and cells were incubated for an additional 12 days. 0 Day , cell size at the time of plating. The sizes of spheres were photographed and measured on a 100 μm scale at a magnification of 400× using an Olympus microscope. Data represent the mean ± standard deviation of 15 sphere determinations. ** P
    Figure Legend Snippet: DCA and LCA induce increases in CSC markers. Quantitative real-time PCR showed significantly increased mRNA expression of CD44 ( a ), CD166 ( b ), and ALDHA1 ( c ) in HCoEpiC following incubation with 100 μM DCA or LCA for 72 h. Flow cytometric analysis of HCoEpiC showing an increased proportion of CD44-positive and CD166-positive cells following 12-day incubation in the absence (control) or presence of 100 μM DCA or LCA ( d ). DCA/LCA mediated the increase in spheroid formation by the CD44 + CD166 – CSC phenotype of HCoEpiC ( e ); photomicrographs showing spheroids formed in response to DCA or LCA at the end of the 12-day incubation period. For spheroid formation, CD44 + CD166 – cells were sorted by flow cytometry and 200–300 cells were seeded with B27 containing DMEM/F12 medium in 96-well low-attachment plates; 24 h after seeding, 100 μM DCA or LCA was added to the incubation medium, and cells were incubated for an additional 12 days. 0 Day , cell size at the time of plating. The sizes of spheres were photographed and measured on a 100 μm scale at a magnification of 400× using an Olympus microscope. Data represent the mean ± standard deviation of 15 sphere determinations. ** P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Incubation, Flow Cytometry, Cytometry, Microscopy, Standard Deviation

    32) Product Images from "EMAP-II sensitize U87MG and glioma stem-like cells to temozolomide via induction of autophagy-mediated cell death and G2/M arrest"

    Article Title: EMAP-II sensitize U87MG and glioma stem-like cells to temozolomide via induction of autophagy-mediated cell death and G2/M arrest

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1315492

    EMAP-II enhanced cytotoxic effect of TMZ on U87MG and GSCs (A) U87MG cells were cultured in the DMEM, containing 10% FBS in flasks. After sorted by FACS, the CD133-positive cells were collected and maintained in the DMEM/F12, containing 2% B27 supplements, 20ng/ml EGF, and bFGF. Suspended cells became spheres after 48 h culture. (B) GSCs expressing the stemness biomarkers of CD133 and Nestin by immunofluorescence. (C) Both U87MG and GSCs were treated with TMZ alone or a combination of TMZ and EMAP-II with assigned concentration for 72 h respectively, IC 50 of TMZ calculated from dose-inhibition response curves was remarkably decreased on both U87MG and GSCs. (D) Cells were treated with 100μM TMZ and/or 0.5 nM EMAP-II for 72 h respectively and cell viability was then determined. EMAP-II enhanced the TMZ-induced cytotoxic effect on U87MG and GSCs.
    Figure Legend Snippet: EMAP-II enhanced cytotoxic effect of TMZ on U87MG and GSCs (A) U87MG cells were cultured in the DMEM, containing 10% FBS in flasks. After sorted by FACS, the CD133-positive cells were collected and maintained in the DMEM/F12, containing 2% B27 supplements, 20ng/ml EGF, and bFGF. Suspended cells became spheres after 48 h culture. (B) GSCs expressing the stemness biomarkers of CD133 and Nestin by immunofluorescence. (C) Both U87MG and GSCs were treated with TMZ alone or a combination of TMZ and EMAP-II with assigned concentration for 72 h respectively, IC 50 of TMZ calculated from dose-inhibition response curves was remarkably decreased on both U87MG and GSCs. (D) Cells were treated with 100μM TMZ and/or 0.5 nM EMAP-II for 72 h respectively and cell viability was then determined. EMAP-II enhanced the TMZ-induced cytotoxic effect on U87MG and GSCs.

    Techniques Used: Cell Culture, FACS, Expressing, Immunofluorescence, Concentration Assay, Inhibition

    33) Product Images from "The dysregulated pharmacology of clinically relevant ESR1 mutants is normalized by ligand-activated WT receptor"

    Article Title: The dysregulated pharmacology of clinically relevant ESR1 mutants is normalized by ligand-activated WT receptor

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-19-1148

    Cells expressing both the ER WT and ER muts have similar pharmacological responses to antiestrogens when compared to cells only expressing ER WT . (A-G) MCF7 B cells were grown in DMEM-F12 media containing 2% FBS for 7 days while being treated with ER antagonists (10 −12 -10 −6 M). Cellular proliferation was assessed by measuring DNA content (Hoechst stain) and DNA content is normalized to vehicle. Data points are the mean of three technical replicates, and error bars are the standard deviation of these replicates. Data presented is a representative of three independent experiments. Two-way ANOVA was utilized, comparing the logIC 50 of all three independent experiments, to determine if there were significant differences between the WT and mutant receptors. No significant differences (p-value
    Figure Legend Snippet: Cells expressing both the ER WT and ER muts have similar pharmacological responses to antiestrogens when compared to cells only expressing ER WT . (A-G) MCF7 B cells were grown in DMEM-F12 media containing 2% FBS for 7 days while being treated with ER antagonists (10 −12 -10 −6 M). Cellular proliferation was assessed by measuring DNA content (Hoechst stain) and DNA content is normalized to vehicle. Data points are the mean of three technical replicates, and error bars are the standard deviation of these replicates. Data presented is a representative of three independent experiments. Two-way ANOVA was utilized, comparing the logIC 50 of all three independent experiments, to determine if there were significant differences between the WT and mutant receptors. No significant differences (p-value

    Techniques Used: Expressing, Staining, Standard Deviation, Mutagenesis, Significance Assay

    34) Product Images from "Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells"

    Article Title: Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-50648-5

    Ki-67 expression is diminished after AM-CM treatment. ( A ) HepG2 cells were seeded in 24-wells plate and treated with DMEM-F12 10% FBS (C), DMEM-F12 0% FBS (0% FBS), AM-CM 50% diluted (AM-CM 1/2), AM-CM 25% diluted (AM-CM 1/4), at indicated times. Cells were fixed and Ki-67 expression (green) was detected using Alexa-488 conjugated secondary antibody, at indicated times. Representative micrographs from HepG2 taken at 10X are shown. The nuclei were stained with DAPI (blue). ( B , C ) Graph shows the number of HepG2 nucleus positive for Ki-67 at 24 h ( B ) and 72 h ( C ) of treatment. Scale Bar: 100 μm. Results are expressed as means ± S.D. (n = 5) and one from a representative experiment is shown. ***p
    Figure Legend Snippet: Ki-67 expression is diminished after AM-CM treatment. ( A ) HepG2 cells were seeded in 24-wells plate and treated with DMEM-F12 10% FBS (C), DMEM-F12 0% FBS (0% FBS), AM-CM 50% diluted (AM-CM 1/2), AM-CM 25% diluted (AM-CM 1/4), at indicated times. Cells were fixed and Ki-67 expression (green) was detected using Alexa-488 conjugated secondary antibody, at indicated times. Representative micrographs from HepG2 taken at 10X are shown. The nuclei were stained with DAPI (blue). ( B , C ) Graph shows the number of HepG2 nucleus positive for Ki-67 at 24 h ( B ) and 72 h ( C ) of treatment. Scale Bar: 100 μm. Results are expressed as means ± S.D. (n = 5) and one from a representative experiment is shown. ***p

    Techniques Used: Expressing, Staining

    HepG2 cells cycle progression is inhibited by AM-CM treatment. HepG2 cells (5 × 10 5 ) were grown in 6-wells plate in DMEM-F12 10% FBS during 24 h. Then, medium was replaced by treatment with DMEM 10% FBS (C), DMEM 0% FBS (0% FBS), AM-CM 1/2 or AM-CM 1/4. After 24 h and 72 h cells were processed and analyzed by flow cytometry, as indicated in Materials and Methods. In ( A , C ) the cell population was selected on the basis of size (FSC-H) and granularity (SSC-H) and this population was plotted in PI-A vs PI-W plot in order to discriminate doublet population. Then singlet population was selected and plotted in count vs PI (propidium iodide) intensity graph. Graphs represent the distribution of HepG2 cells in different phases of cell cycle at 24 h ( A ) or 72 h ( C ) of treatment. The peak on the left in all graphs represents the G1 population and peak on the right represents the G2/M population. The valley between these two peaks represents the S phase population ( B – D ). The bar diagram represented the % of cells in different phases of cell cycle after 24 h ( B ) or 72 h ( D ) of treatment. Results are expressed as means ± S.D. (n = 3) and one from a representative experiment is shown. *p
    Figure Legend Snippet: HepG2 cells cycle progression is inhibited by AM-CM treatment. HepG2 cells (5 × 10 5 ) were grown in 6-wells plate in DMEM-F12 10% FBS during 24 h. Then, medium was replaced by treatment with DMEM 10% FBS (C), DMEM 0% FBS (0% FBS), AM-CM 1/2 or AM-CM 1/4. After 24 h and 72 h cells were processed and analyzed by flow cytometry, as indicated in Materials and Methods. In ( A , C ) the cell population was selected on the basis of size (FSC-H) and granularity (SSC-H) and this population was plotted in PI-A vs PI-W plot in order to discriminate doublet population. Then singlet population was selected and plotted in count vs PI (propidium iodide) intensity graph. Graphs represent the distribution of HepG2 cells in different phases of cell cycle at 24 h ( A ) or 72 h ( C ) of treatment. The peak on the left in all graphs represents the G1 population and peak on the right represents the G2/M population. The valley between these two peaks represents the S phase population ( B – D ). The bar diagram represented the % of cells in different phases of cell cycle after 24 h ( B ) or 72 h ( D ) of treatment. Results are expressed as means ± S.D. (n = 3) and one from a representative experiment is shown. *p

    Techniques Used: Flow Cytometry

    Pro and antitumoral miRs expression is modulated by AM-CM in HuH-7 cells. ( A – D ) HuH-7 cells were plated in complete DMEM-F12 medium supplemented with 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h (left) or 72 h (right) before RNA extraction. Total RNA was extracted as described in Materials and Methods. Relative expression of miR-15a ( A ), miR-210 ( B ), miR-206 ( C ), miR-145 ( D ) was determined by q RT-PCR. RNU6B was used as internal control for normalization. *p
    Figure Legend Snippet: Pro and antitumoral miRs expression is modulated by AM-CM in HuH-7 cells. ( A – D ) HuH-7 cells were plated in complete DMEM-F12 medium supplemented with 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h (left) or 72 h (right) before RNA extraction. Total RNA was extracted as described in Materials and Methods. Relative expression of miR-15a ( A ), miR-210 ( B ), miR-206 ( C ), miR-145 ( D ) was determined by q RT-PCR. RNU6B was used as internal control for normalization. *p

    Techniques Used: Expressing, Incubation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

    AM-CM induces p21 expression and translocation to HepG2 cells nucleus. HepG2 cells were plated in complete DMEM-F12 medium supplemented with 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h ( A ) or 72 h ( B ) before RNA extraction. Total RNA was extracted as described in Materials and Methods. P21 mRNA was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C ) HepG2 cells were seeded in 24-wells plate and incubated in DMEM-F12 10% FBS (C), DMEM-F12 0% FBS, AM-CM 50% dilution (AM-CM 1/2), or AM-CM 25% dilution (AM-CM 1/4), during indicated times. P21 expression (green) was detected in fixed cells using Alexa-488 conjugated secondary antibody. Representative micrographs at 10X from HepG2 at 24 h and 72 h are shown. The nuclei were stained with DAPI (blue). ( D ) Graph shows quantification of nuclear p21 fluorescence intensity in HepG2 cells. Scale Bar: 100 μm. ( E ) Graph shows p21 nuclear localization related to cytoplasmic localization after AM-CM 1/2 and AM-CM 1/4 treatments. Results are expressed as mean ± S.D. for five independent experiments. *p
    Figure Legend Snippet: AM-CM induces p21 expression and translocation to HepG2 cells nucleus. HepG2 cells were plated in complete DMEM-F12 medium supplemented with 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h ( A ) or 72 h ( B ) before RNA extraction. Total RNA was extracted as described in Materials and Methods. P21 mRNA was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C ) HepG2 cells were seeded in 24-wells plate and incubated in DMEM-F12 10% FBS (C), DMEM-F12 0% FBS, AM-CM 50% dilution (AM-CM 1/2), or AM-CM 25% dilution (AM-CM 1/4), during indicated times. P21 expression (green) was detected in fixed cells using Alexa-488 conjugated secondary antibody. Representative micrographs at 10X from HepG2 at 24 h and 72 h are shown. The nuclei were stained with DAPI (blue). ( D ) Graph shows quantification of nuclear p21 fluorescence intensity in HepG2 cells. Scale Bar: 100 μm. ( E ) Graph shows p21 nuclear localization related to cytoplasmic localization after AM-CM 1/2 and AM-CM 1/4 treatments. Results are expressed as mean ± S.D. for five independent experiments. *p

    Techniques Used: Expressing, Translocation Assay, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction, Staining, Fluorescence

    Pro and antitumoral miRs expression is modulated by AM-CM in HepG2 cells. ( A , D ) HepG2 cells were plated in complete DMEM-F12 medium 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h (left) or 72 h (right) before RNA extraction. Relative expression of miR-15a ( A ), miR-210 ( B ), miR-206 ( C ), miR-145 ( D ) was determined by qRT-PCR. RNU6B was used as internal control for normalization. *p
    Figure Legend Snippet: Pro and antitumoral miRs expression is modulated by AM-CM in HepG2 cells. ( A , D ) HepG2 cells were plated in complete DMEM-F12 medium 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h (left) or 72 h (right) before RNA extraction. Relative expression of miR-15a ( A ), miR-210 ( B ), miR-206 ( C ), miR-145 ( D ) was determined by qRT-PCR. RNU6B was used as internal control for normalization. *p

    Techniques Used: Expressing, Incubation, RNA Extraction, Quantitative RT-PCR

    Key proliferation proteins expression is downregulated by AM-CM treatment. HepG2 cells were incubated with DMEM-F12 10% FBS (C), DMEM-F12 0% FBS, AM-CM 50% diluted (AM-CM 1/2), AM-CM 25% diluted (AM-CM 1/4) during 24 ( A ) or 72 h ( B ) before RNA extraction. CYCLIN D1 mRNA was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C , D ) HepG2 cells were seeded in 10-cm plate and incubated with complete DMEM-F12 medium supplemented with 10% FBS (C), or without serum (0% FBS), or with AM-CM 50% (AM-CM 1/2) or AM-CM 25% (AM-CM 1/4). After 24 h ( C ) or 72 h ( D ). Cyclin D1 expression in cell extracts was determined by Western blot. Molecular weights were estimated using standard protein markers. Loading controls were performed by immunoblotting the same membranes with anti-GAPDH. Lower panels show bands densitometry. Full-length blots are available in Supplementary Dataset. Molecular weight (kDa) is indicated at the right of the blot. *p
    Figure Legend Snippet: Key proliferation proteins expression is downregulated by AM-CM treatment. HepG2 cells were incubated with DMEM-F12 10% FBS (C), DMEM-F12 0% FBS, AM-CM 50% diluted (AM-CM 1/2), AM-CM 25% diluted (AM-CM 1/4) during 24 ( A ) or 72 h ( B ) before RNA extraction. CYCLIN D1 mRNA was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C , D ) HepG2 cells were seeded in 10-cm plate and incubated with complete DMEM-F12 medium supplemented with 10% FBS (C), or without serum (0% FBS), or with AM-CM 50% (AM-CM 1/2) or AM-CM 25% (AM-CM 1/4). After 24 h ( C ) or 72 h ( D ). Cyclin D1 expression in cell extracts was determined by Western blot. Molecular weights were estimated using standard protein markers. Loading controls were performed by immunoblotting the same membranes with anti-GAPDH. Lower panels show bands densitometry. Full-length blots are available in Supplementary Dataset. Molecular weight (kDa) is indicated at the right of the blot. *p

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction, Western Blot, Molecular Weight

    P53 expression increases in HepG2 cells after AM-CM treatment. HepG2 cells were plated in complete DMEM-F12 medium 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2) or at 25% (AM-CM 1/4) and incubated during 24 h ( A ) or 72 h ( B ) before total RNA extraction. P53 mRNA was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C , D ) HepG2 cells were seeded in 10-cm plate and incubated with complete DMEM-F12 10% FBS (C), or without serum (0% FBS), or with AM-CM 50% (AM-CM 1/2) or AM-CM 25% (AM-CM 1/4). Cell extracts were prepared at indicated times and proteins were separated on SDS-PAGE gels. P53 expression at 24 h ( C ) or 72 h after treatment ( D ) was determined by Western blot. Molecular weight was estimated using standard protein markers and is indicated at the right of the blot. Loading controls were performed by GAPDH detection. Bands densitometry is shown in lower panels. Full-length blots are available in Supplementary Dataset. Results from a representative experiment are shown and expressed as means ± S.D. for five independent experiments performed in duplicates. For Western blot, representative results from three replicates are shown. *p
    Figure Legend Snippet: P53 expression increases in HepG2 cells after AM-CM treatment. HepG2 cells were plated in complete DMEM-F12 medium 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2) or at 25% (AM-CM 1/4) and incubated during 24 h ( A ) or 72 h ( B ) before total RNA extraction. P53 mRNA was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C , D ) HepG2 cells were seeded in 10-cm plate and incubated with complete DMEM-F12 10% FBS (C), or without serum (0% FBS), or with AM-CM 50% (AM-CM 1/2) or AM-CM 25% (AM-CM 1/4). Cell extracts were prepared at indicated times and proteins were separated on SDS-PAGE gels. P53 expression at 24 h ( C ) or 72 h after treatment ( D ) was determined by Western blot. Molecular weight was estimated using standard protein markers and is indicated at the right of the blot. Loading controls were performed by GAPDH detection. Bands densitometry is shown in lower panels. Full-length blots are available in Supplementary Dataset. Results from a representative experiment are shown and expressed as means ± S.D. for five independent experiments performed in duplicates. For Western blot, representative results from three replicates are shown. *p

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Molecular Weight

    AM-CM inhibits hepatocarcinoma cells proliferation and viability. ( A ) After HepG2 cells treatment with DMEM 10% FBS (C), DMEM 0% FBS (0% FBS) or AM-CM pure, cell lysates were prepared and 3 H-thymidine incorporation was determined as described in Materials and Methods. HepG2 cells ( B , D ) and HuH-7 cells ( C , E ) were seeded in 24-wells plate in complete DMEM medium supplemented with 10% FBS (C) or in DMEM 0% FBS (0% FBS) or in AM-CM pure (AM-CM), diluted at 50% (AM-CM 1/2) or diluted at 25% (AM-CM 1/4); or with AM-CM 1/2 10% FBS or AM-CM 1/4 10% FBS ( D , E ). Cells viability was determined by MTT test at 24, 48 and 72 h of culture. HepG2 cells ( F ) or HuH-7 cells ( G ) were seeded in 24-wells plate and before AM-CM treatment were exposed to 40 J/m 2 of UVC (5 sec). After exposure, cells were grown in DMEM-F12 10% FBS (C), DMEM-F12 0% FBS ( UV -0% FBS) or treated with AM-CM pure ( UV -AM-CM), 50% diluted ( UV -AM-CM 1/2) or 25% diluted ( UV -AM-CM 1/4). Cell viability was determined by MTT test at indicated times. In control cells with DMEM-F12 10% FBS, UV treatment was omitted. Independent experiments were performed in duplicates five times. Results are expressed as means ± S.D. *p
    Figure Legend Snippet: AM-CM inhibits hepatocarcinoma cells proliferation and viability. ( A ) After HepG2 cells treatment with DMEM 10% FBS (C), DMEM 0% FBS (0% FBS) or AM-CM pure, cell lysates were prepared and 3 H-thymidine incorporation was determined as described in Materials and Methods. HepG2 cells ( B , D ) and HuH-7 cells ( C , E ) were seeded in 24-wells plate in complete DMEM medium supplemented with 10% FBS (C) or in DMEM 0% FBS (0% FBS) or in AM-CM pure (AM-CM), diluted at 50% (AM-CM 1/2) or diluted at 25% (AM-CM 1/4); or with AM-CM 1/2 10% FBS or AM-CM 1/4 10% FBS ( D , E ). Cells viability was determined by MTT test at 24, 48 and 72 h of culture. HepG2 cells ( F ) or HuH-7 cells ( G ) were seeded in 24-wells plate and before AM-CM treatment were exposed to 40 J/m 2 of UVC (5 sec). After exposure, cells were grown in DMEM-F12 10% FBS (C), DMEM-F12 0% FBS ( UV -0% FBS) or treated with AM-CM pure ( UV -AM-CM), 50% diluted ( UV -AM-CM 1/2) or 25% diluted ( UV -AM-CM 1/4). Cell viability was determined by MTT test at indicated times. In control cells with DMEM-F12 10% FBS, UV treatment was omitted. Independent experiments were performed in duplicates five times. Results are expressed as means ± S.D. *p

    Techniques Used: MTT Assay

    Mdm-2 expression decreases in HepG2 cells after AM-CM treatment. HepG2 cells were plated in DMEM-F12 medium supplemented with 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h ( A ) or 72 h ( B ) before RNA extraction. MDM-2 mRNA expression was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C – E ) HepG2 cells incubated in DMEM-F12 10% FBS (C), DMEM-F12 0% FBS, AM-CM 50% dilution (AM-CM 1/2), or AM-CM 25% dilution (AM-CM 1/4), during indicated times. Cells were fixed at specified times and Mdm-2 expression (green) was detected using Alexa-488 conjugated secondary antibody. Representative micrographs at 60X from HepG2 cells at 24 h ( C ) and 72 h ( D ) are shown. The nuclei were stained with DAPI (blue). Graphs below show Mdm-2 fluorescence intensity in HepG2 cells at 24 h ( E ) and 72 h ( F ) of treatment. Scale Bar: 8 μm. Results are expressed as mean ± S.D. (n = 5). **p
    Figure Legend Snippet: Mdm-2 expression decreases in HepG2 cells after AM-CM treatment. HepG2 cells were plated in DMEM-F12 medium supplemented with 10% FBS (C), in DMEM-F12 0% FBS (0% FBS), in AM-CM diluted at 50% (AM-CM 1/2), AM-CM at 25% (AM-CM 1/4) and incubated during 24 h ( A ) or 72 h ( B ) before RNA extraction. MDM-2 mRNA expression was measured by quantitative real time PCR. CYCLOPHILIN and GAPDH were used as internal standards. ( C – E ) HepG2 cells incubated in DMEM-F12 10% FBS (C), DMEM-F12 0% FBS, AM-CM 50% dilution (AM-CM 1/2), or AM-CM 25% dilution (AM-CM 1/4), during indicated times. Cells were fixed at specified times and Mdm-2 expression (green) was detected using Alexa-488 conjugated secondary antibody. Representative micrographs at 60X from HepG2 cells at 24 h ( C ) and 72 h ( D ) are shown. The nuclei were stained with DAPI (blue). Graphs below show Mdm-2 fluorescence intensity in HepG2 cells at 24 h ( E ) and 72 h ( F ) of treatment. Scale Bar: 8 μm. Results are expressed as mean ± S.D. (n = 5). **p

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction, Staining, Fluorescence

    35) Product Images from "The Effect of Inflammatory Priming on the Therapeutic Potential of Mesenchymal Stromal Cells for Spinal Cord Repair"

    Article Title: The Effect of Inflammatory Priming on the Therapeutic Potential of Mesenchymal Stromal Cells for Spinal Cord Repair

    Journal: Cells

    doi: 10.3390/cells10061316

    Experimental design. ( A ) The bone marrow was extracted from rats’ femurs and tibias and cultured on plastic dishes for 24 h. Then, monocytes were sorted using FACS and cultured for 7 days until differentiated to bone-marrow-derived macrophages. Mature macrophages were polarized to pro-inflammatory cells by culture in medium with LPS and IFNγ for 24 h. The conditioned medium (CM) from pro-inflammatory macrophages was collected and, later, used to prime MSC. In parallel, bone-marrow-derived MSC, adhered to the dish during the 24 h of bone marrow culture, were maintained in culture until passage 2, then FACS-sorted for purification. Sorted MSC were transduced with GFP-lentivirus to turn them green and trackable. P4 GFP-positive MSC were divided into two batches; one was cultured for 24 h in pro-inflammatory macrophage CM (primed MSC, pMSC) and the other one was cultured for 24 h in fresh D10 (naïve MSC, nMSC). pMSC and nMSC were trypsinized and resuspended in DMEM:F12 for transplantation. ( B ) Rats were acclimated to the behavioral tests before surgery, and the baseline scores for each test were obtained. Surgery for contusive SCI was performed. and the next day, BBB scores of the injured rats were collected. Three days after injury, surgery for direct injection of pMSC, nMSC, or DMEM:F12 was performed. At 1 and 4 wpt, six rats per group and time point were fixed and their spinal cords dissected for histological analysis. Ten additional rats per group were kept alive for 6 wpt, and their sensorimotor performance was measured using BBB scores weekly after transplant, the horizontal ladder test at 3 and 6 wpt, and the Von Frey test at 4 and 6 wpt. At 6 wpt, those rats were fixed and their spinal cords dissected for histological analysis. Abbreviations: MSC: mesenchymal stromal cell; P2: passage 2; LPS: lipopolysaccharide; IFNγ: interferon-gamma; GFP: green fluorescent protein; LV: lentivirus; D10: DMEM medium with 10% fetal bovine serum and 0.1% gentamycin; SCI: spinal cord injury; BBB: Basso, Beattie, Bresnahan locomotor scale; wpt: weeks post-transplant.
    Figure Legend Snippet: Experimental design. ( A ) The bone marrow was extracted from rats’ femurs and tibias and cultured on plastic dishes for 24 h. Then, monocytes were sorted using FACS and cultured for 7 days until differentiated to bone-marrow-derived macrophages. Mature macrophages were polarized to pro-inflammatory cells by culture in medium with LPS and IFNγ for 24 h. The conditioned medium (CM) from pro-inflammatory macrophages was collected and, later, used to prime MSC. In parallel, bone-marrow-derived MSC, adhered to the dish during the 24 h of bone marrow culture, were maintained in culture until passage 2, then FACS-sorted for purification. Sorted MSC were transduced with GFP-lentivirus to turn them green and trackable. P4 GFP-positive MSC were divided into two batches; one was cultured for 24 h in pro-inflammatory macrophage CM (primed MSC, pMSC) and the other one was cultured for 24 h in fresh D10 (naïve MSC, nMSC). pMSC and nMSC were trypsinized and resuspended in DMEM:F12 for transplantation. ( B ) Rats were acclimated to the behavioral tests before surgery, and the baseline scores for each test were obtained. Surgery for contusive SCI was performed. and the next day, BBB scores of the injured rats were collected. Three days after injury, surgery for direct injection of pMSC, nMSC, or DMEM:F12 was performed. At 1 and 4 wpt, six rats per group and time point were fixed and their spinal cords dissected for histological analysis. Ten additional rats per group were kept alive for 6 wpt, and their sensorimotor performance was measured using BBB scores weekly after transplant, the horizontal ladder test at 3 and 6 wpt, and the Von Frey test at 4 and 6 wpt. At 6 wpt, those rats were fixed and their spinal cords dissected for histological analysis. Abbreviations: MSC: mesenchymal stromal cell; P2: passage 2; LPS: lipopolysaccharide; IFNγ: interferon-gamma; GFP: green fluorescent protein; LV: lentivirus; D10: DMEM medium with 10% fetal bovine serum and 0.1% gentamycin; SCI: spinal cord injury; BBB: Basso, Beattie, Bresnahan locomotor scale; wpt: weeks post-transplant.

    Techniques Used: Cell Culture, FACS, Derivative Assay, Purification, Transduction, Transplantation Assay, Injection

    36) Product Images from "Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases"

    Article Title: Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2022.101510

    Lalistat 2 (L2) does not affect adipocyte differentiation but reduces isoproterenol-stimulated lipolysis. Stromal vascular cells were isolated from subcutaneous white adipose tissue of wild-type mice and differentiated into mature adipocytes in the absence (- L2) or presence of 30 μM Lalistat 2 (+ L2); 0.02% DMSO was used as control. (A) Acid (pH 4.5) cholesteryl ester hydrolase (CEH) and (B) triglyceride hydrolase activity (TGH) activity in lysates of adipocytes in the absence (- L2 in lysate) or presence (+ L2 in lysate) of 30 μM L2. (C) Lipid staining with oil red O (magnification, 10x; scale bar, 100 μm) and (D) quantification of TG, total cholesterol (TC), and CE concentrations. (E) mRNA expression of genes associated with lipid metabolism. (F) Basal and isoproterenol (10 μM) stimulated lipolysis in adipocytes determined as FA release. (G) Neutral (pH 7) TGH activity of adipocytes +/− 30 μM L2 in differentiation medium and lysates. Data (n = 3) represent mean + SD. The results are representative of two independent experiments. Statistically significant differences were calculated by (D, E) Student's t-test or (A, B, F, G) 1-way ANOVA followed by Bonferroni post-hoc test; ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 for comparisons within the group (−/+ L2); ## p ≤ 0.01, ### p ≤ 0.001 for comparisons between the groups (−/+ L2 in lysate or basal/isoproterenol-stimulated).
    Figure Legend Snippet: Lalistat 2 (L2) does not affect adipocyte differentiation but reduces isoproterenol-stimulated lipolysis. Stromal vascular cells were isolated from subcutaneous white adipose tissue of wild-type mice and differentiated into mature adipocytes in the absence (- L2) or presence of 30 μM Lalistat 2 (+ L2); 0.02% DMSO was used as control. (A) Acid (pH 4.5) cholesteryl ester hydrolase (CEH) and (B) triglyceride hydrolase activity (TGH) activity in lysates of adipocytes in the absence (- L2 in lysate) or presence (+ L2 in lysate) of 30 μM L2. (C) Lipid staining with oil red O (magnification, 10x; scale bar, 100 μm) and (D) quantification of TG, total cholesterol (TC), and CE concentrations. (E) mRNA expression of genes associated with lipid metabolism. (F) Basal and isoproterenol (10 μM) stimulated lipolysis in adipocytes determined as FA release. (G) Neutral (pH 7) TGH activity of adipocytes +/− 30 μM L2 in differentiation medium and lysates. Data (n = 3) represent mean + SD. The results are representative of two independent experiments. Statistically significant differences were calculated by (D, E) Student's t-test or (A, B, F, G) 1-way ANOVA followed by Bonferroni post-hoc test; ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 for comparisons within the group (−/+ L2); ## p ≤ 0.01, ### p ≤ 0.001 for comparisons between the groups (−/+ L2 in lysate or basal/isoproterenol-stimulated).

    Techniques Used: Isolation, Mouse Assay, Activity Assay, Staining, Expressing

    37) Product Images from "Adamantyl Retinoid-Related Molecules Induce Apoptosis in Pancreatic Cancer Cells by Inhibiting IGF-1R and Wnt/β-Catenin Pathways"

    Article Title: Adamantyl Retinoid-Related Molecules Induce Apoptosis in Pancreatic Cancer Cells by Inhibiting IGF-1R and Wnt/β-Catenin Pathways

    Journal: Journal of Oncology

    doi: 10.1155/2012/796729

    3-Cl-AHPC-mediated inhibition and degradation of pancreatic cancer stem cells spheres of CD133 + , CD44 + /CD24 + /EpCAM + , and stem-like spheres of CD44 + /CD24 + PANC-1 cells. ((a), (b) and (c), (d)) 3-Cl-AHPC and AHP3 exposure resulted in inhibition of CD44 + /CD24 + /EpCAM + and CD133 + cells growth and sphere formation and degradation of the derived spheres. ((e), (f)) AHP3 and 3-Cl-AHPC inhibited sphere formation and inhibition of growth and degradation of the CD44 + /CD24 + -derived spheres. For sphere formation, the CD44 + /CD24 + /EpCAM + , CD133 + , and CD44 + /CD24 + cells were sorted by flow cytometry and approximately 200–300 cells were seeded with B27 containing DMEM/F12 medium in 96-well low attachment plates and 1 μ M 3-Cl-AHPC or AHP3 added either the day after seeding or 7 days following sphere formation. The sizes of spheres were photographed and measured on a 100 μ m scale and magnification 400X using Olympus fluorescence microscope digital camera software and DP2-BSW software. The error bars represent the mean of 15 sphere determinations ± the standard deviation. ** was significantly different in comparison to control spheres. Data were analyzed by ANOVA, Tukey HSD test for multiple comparisons. ** P
    Figure Legend Snippet: 3-Cl-AHPC-mediated inhibition and degradation of pancreatic cancer stem cells spheres of CD133 + , CD44 + /CD24 + /EpCAM + , and stem-like spheres of CD44 + /CD24 + PANC-1 cells. ((a), (b) and (c), (d)) 3-Cl-AHPC and AHP3 exposure resulted in inhibition of CD44 + /CD24 + /EpCAM + and CD133 + cells growth and sphere formation and degradation of the derived spheres. ((e), (f)) AHP3 and 3-Cl-AHPC inhibited sphere formation and inhibition of growth and degradation of the CD44 + /CD24 + -derived spheres. For sphere formation, the CD44 + /CD24 + /EpCAM + , CD133 + , and CD44 + /CD24 + cells were sorted by flow cytometry and approximately 200–300 cells were seeded with B27 containing DMEM/F12 medium in 96-well low attachment plates and 1 μ M 3-Cl-AHPC or AHP3 added either the day after seeding or 7 days following sphere formation. The sizes of spheres were photographed and measured on a 100 μ m scale and magnification 400X using Olympus fluorescence microscope digital camera software and DP2-BSW software. The error bars represent the mean of 15 sphere determinations ± the standard deviation. ** was significantly different in comparison to control spheres. Data were analyzed by ANOVA, Tukey HSD test for multiple comparisons. ** P

    Techniques Used: Inhibition, Derivative Assay, Flow Cytometry, Fluorescence, Microscopy, Software, Standard Deviation

    38) Product Images from "Co-expression of CXCR4 and CXCR7 in human endometrial stromal cells is modulated by steroid hormones"

    Article Title: Co-expression of CXCR4 and CXCR7 in human endometrial stromal cells is modulated by steroid hormones

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Effects of estrogen and progesterone on CXCL12, CXCR4 and CXCR7 mRNA expression in human ESCs. ESCs in mid-log growth were starved with DMEM/F12 for 12 h, then treated with vehicle (DMEM/F12 with 2% stripped FCS), estrogen (10 -11 -10 -6 M) or progesterone
    Figure Legend Snippet: Effects of estrogen and progesterone on CXCL12, CXCR4 and CXCR7 mRNA expression in human ESCs. ESCs in mid-log growth were starved with DMEM/F12 for 12 h, then treated with vehicle (DMEM/F12 with 2% stripped FCS), estrogen (10 -11 -10 -6 M) or progesterone

    Techniques Used: Expressing

    Effects of estrogen and progesterone on membrane expression of CXCR4 and CXCR7 in human ESCs. ESCs in mid-log growth were starved with DMEM/F12 for 12 h, then added with vehicle (DMEM/F12 with 2% stripped FCS), estrogen (10 -9 M), progesterone (10 -10 M)
    Figure Legend Snippet: Effects of estrogen and progesterone on membrane expression of CXCR4 and CXCR7 in human ESCs. ESCs in mid-log growth were starved with DMEM/F12 for 12 h, then added with vehicle (DMEM/F12 with 2% stripped FCS), estrogen (10 -9 M), progesterone (10 -10 M)

    Techniques Used: Expressing

    Modulation of estrogen and progesterone on CXCR4 and CXCR7 protein expression in human ESCs by in-cell Western analysis. ESCs in mid-log growth were starved with DMEM/F12 for 12 h, then treated with vehicle (DMEM/F12 with 2% stripped FCS), estrogen (10
    Figure Legend Snippet: Modulation of estrogen and progesterone on CXCR4 and CXCR7 protein expression in human ESCs by in-cell Western analysis. ESCs in mid-log growth were starved with DMEM/F12 for 12 h, then treated with vehicle (DMEM/F12 with 2% stripped FCS), estrogen (10

    Techniques Used: Expressing, In-Cell ELISA

    39) Product Images from "Intracellularly Released Cholesterol from Polymer-Based Delivery Systems Alters Cellular Responses to Pneumolysin and Promotes Cell Survival"

    Article Title: Intracellularly Released Cholesterol from Polymer-Based Delivery Systems Alters Cellular Responses to Pneumolysin and Promotes Cell Survival

    Journal: Metabolites

    doi: 10.3390/metabo11120821

    Intracellular delivery of cholesterol by polymeric particles. ( a ) HepG2 cells were treated with (50 µg mL −1 ) polymeric particles prepared by nano- and microprecipitation, or equimolar amounts of cholesterol dissolved in methanol (MeOH) or methyl-β-cyclodextrin (MCD) in DMEM:F12. The change in cellular un-esterified cholesterol is depicted as log 2 -fold relative to the cholesterol level of untreated control cells at 1.5 h; means and standard deviations are provided in Supplementary Table S3 . ( b ) Distribution of cholesterol was assessed by fluorescence microscopy of filipin III stained HepG2 cells following 1.5 h stimulation with [E100–PLGA](Chol) NP and [E100–PLGA] NP . The contrast has been adjusted equally on the depicted images for better visualization. Staining intensity does not reflect cholesterol levels as measured by mass spectrometry. ( c ) Cholesterol-rich vesicles (yellow dots) were identified in proximity to the plasma membrane by thresholding filipin III fluorescence images. The number of cholesterol-containing vesicles per cell was calculated after cell nuclei were counted. Feret’s diameter of the identified vesicles was examined by applying the analyzed particle function in the Fiji distribution of ImageJ. Supplementation of the nanocarrier significantly increases the number of cholesterol-rich vesicles per cell, as shown in Tukey’s box plot. ( d ) Inhibition of cellular uptake by 25 µmol L −1 PitStop-2 (PS-2) before 3 h stimulation with [E100–PLGA](Chol) NP (50 µg mL −1 ) reduced protective effects of cholesterol supplementation as measured by LDH release assay. * p
    Figure Legend Snippet: Intracellular delivery of cholesterol by polymeric particles. ( a ) HepG2 cells were treated with (50 µg mL −1 ) polymeric particles prepared by nano- and microprecipitation, or equimolar amounts of cholesterol dissolved in methanol (MeOH) or methyl-β-cyclodextrin (MCD) in DMEM:F12. The change in cellular un-esterified cholesterol is depicted as log 2 -fold relative to the cholesterol level of untreated control cells at 1.5 h; means and standard deviations are provided in Supplementary Table S3 . ( b ) Distribution of cholesterol was assessed by fluorescence microscopy of filipin III stained HepG2 cells following 1.5 h stimulation with [E100–PLGA](Chol) NP and [E100–PLGA] NP . The contrast has been adjusted equally on the depicted images for better visualization. Staining intensity does not reflect cholesterol levels as measured by mass spectrometry. ( c ) Cholesterol-rich vesicles (yellow dots) were identified in proximity to the plasma membrane by thresholding filipin III fluorescence images. The number of cholesterol-containing vesicles per cell was calculated after cell nuclei were counted. Feret’s diameter of the identified vesicles was examined by applying the analyzed particle function in the Fiji distribution of ImageJ. Supplementation of the nanocarrier significantly increases the number of cholesterol-rich vesicles per cell, as shown in Tukey’s box plot. ( d ) Inhibition of cellular uptake by 25 µmol L −1 PitStop-2 (PS-2) before 3 h stimulation with [E100–PLGA](Chol) NP (50 µg mL −1 ) reduced protective effects of cholesterol supplementation as measured by LDH release assay. * p

    Techniques Used: Fluorescence, Microscopy, Staining, Mass Spectrometry, Inhibition, Lactate Dehydrogenase Assay

    Evaluation of cell toxicity after polymer particle stimulation or pneumolysin stress. ( a ) HepG2 cells were treated with polymeric particles prepared by nano- or microprecipitation (NP or MP, respectively) of indicated concentrations in DMEM:F12 for 3 h. Stripes indicate the cholesterol cargo of the polymer particle. Mean diameter size (rounded) of tested sample and polymer composition ( b ) after lyophilization is shown. The polymer surface was coated with poly(2-oxazoline) (POx). ( c ) Cells were challenged with PLY (250 ng mL −1 ) for 3 h in the presence of nano- or microprecipitated particles (50 µg mL −1 ) before toxicity was examined by the release of cytoplasmic LDH. ( d ) Amelioration of PLY toxicity by [E100–PLGA](Chol) NP was found to be time-dependent. Mean ± SEM toxicity is shown relative to completely lysed cells (= 100%). * p
    Figure Legend Snippet: Evaluation of cell toxicity after polymer particle stimulation or pneumolysin stress. ( a ) HepG2 cells were treated with polymeric particles prepared by nano- or microprecipitation (NP or MP, respectively) of indicated concentrations in DMEM:F12 for 3 h. Stripes indicate the cholesterol cargo of the polymer particle. Mean diameter size (rounded) of tested sample and polymer composition ( b ) after lyophilization is shown. The polymer surface was coated with poly(2-oxazoline) (POx). ( c ) Cells were challenged with PLY (250 ng mL −1 ) for 3 h in the presence of nano- or microprecipitated particles (50 µg mL −1 ) before toxicity was examined by the release of cytoplasmic LDH. ( d ) Amelioration of PLY toxicity by [E100–PLGA](Chol) NP was found to be time-dependent. Mean ± SEM toxicity is shown relative to completely lysed cells (= 100%). * p

    Techniques Used:

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    FasL is generated in Rac1-expressing cells after serum deprivation and it is necessary for apoptosis. (A) <t>NIH</t> 3T3 cells stably expressing Ras-1QL protein (Rac1) or control NIH 3T3 cells (Control) were incubated in <t>DMEM</t> with (+) or without (−) serum for 24 h. Then, cells were processed for FasL expression determined by RT-PCR as described in MATERIALS AND METHODS. FasL expression in shown on the left (Fas Ligand), and as control, the β-actin expression (β-Actin) was determined with the same samples in parallel reactions. Negative control refers to an RT-PCR amplification without primers. (B) Western blot analysis of FasL levels in NIH 3T3 and Rac1 cells in the presence and absence of serum. (C) Cells were incubated with FasFc (0.2 μg/ml) either in control medium or without serum for 24 h. Cell death was estimated by flow cytometry (Annexin V) and expressed as percentage of the value of Rac1 without serum for each experiment (cell death range: 22–38%). Data are the mean ± SD from three independent experiments.
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    FasL is generated in Rac1-expressing cells after serum deprivation and it is necessary for apoptosis. (A) NIH 3T3 cells stably expressing Ras-1QL protein (Rac1) or control NIH 3T3 cells (Control) were incubated in DMEM with (+) or without (−) serum for 24 h. Then, cells were processed for FasL expression determined by RT-PCR as described in MATERIALS AND METHODS. FasL expression in shown on the left (Fas Ligand), and as control, the β-actin expression (β-Actin) was determined with the same samples in parallel reactions. Negative control refers to an RT-PCR amplification without primers. (B) Western blot analysis of FasL levels in NIH 3T3 and Rac1 cells in the presence and absence of serum. (C) Cells were incubated with FasFc (0.2 μg/ml) either in control medium or without serum for 24 h. Cell death was estimated by flow cytometry (Annexin V) and expressed as percentage of the value of Rac1 without serum for each experiment (cell death range: 22–38%). Data are the mean ± SD from three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

    doi:

    Figure Lengend Snippet: FasL is generated in Rac1-expressing cells after serum deprivation and it is necessary for apoptosis. (A) NIH 3T3 cells stably expressing Ras-1QL protein (Rac1) or control NIH 3T3 cells (Control) were incubated in DMEM with (+) or without (−) serum for 24 h. Then, cells were processed for FasL expression determined by RT-PCR as described in MATERIALS AND METHODS. FasL expression in shown on the left (Fas Ligand), and as control, the β-actin expression (β-Actin) was determined with the same samples in parallel reactions. Negative control refers to an RT-PCR amplification without primers. (B) Western blot analysis of FasL levels in NIH 3T3 and Rac1 cells in the presence and absence of serum. (C) Cells were incubated with FasFc (0.2 μg/ml) either in control medium or without serum for 24 h. Cell death was estimated by flow cytometry (Annexin V) and expressed as percentage of the value of Rac1 without serum for each experiment (cell death range: 22–38%). Data are the mean ± SD from three independent experiments.

    Article Snippet: NIH 3T3 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% newborn calf serum (NCS) under standard conditions of temperature (37°C), humidity (95°C), and carbon dioxide (5%).

    Techniques: Generated, Expressing, Stable Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Amplification, Western Blot, Flow Cytometry, Cytometry

    Induction of apoptosis by exogenous addition of FasL and/or synthetic ceramides. Analysis of DNA fragmentation was performed essentially as described in MATERIALS AND METHODS. Control, NIH 3T3 cells, and NIH 3T3 overexpressing Rac1 were incubated in DMEM supplemented with 10% NCS until they reached 70% confluence. At this time, cells were incubated in DMEM with (+) or without (−) serum, and treated as follows. (A) Cells were incubated with Fas ligand (50 ng/ml) in the absence or presence of serum for 19 h. (B) Cells were incubated with C 2 ceramide (100 μM) in the presence or absence of serum for 48 h. (C) Cells were incubated with Fas ligand (50 ng/ml) plus ceramides C 2 (100 μM) in the presence or absence of 10% NCS for 24 h.

    Journal: Molecular Biology of the Cell

    Article Title: Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

    doi:

    Figure Lengend Snippet: Induction of apoptosis by exogenous addition of FasL and/or synthetic ceramides. Analysis of DNA fragmentation was performed essentially as described in MATERIALS AND METHODS. Control, NIH 3T3 cells, and NIH 3T3 overexpressing Rac1 were incubated in DMEM supplemented with 10% NCS until they reached 70% confluence. At this time, cells were incubated in DMEM with (+) or without (−) serum, and treated as follows. (A) Cells were incubated with Fas ligand (50 ng/ml) in the absence or presence of serum for 19 h. (B) Cells were incubated with C 2 ceramide (100 μM) in the presence or absence of serum for 48 h. (C) Cells were incubated with Fas ligand (50 ng/ml) plus ceramides C 2 (100 μM) in the presence or absence of 10% NCS for 24 h.

    Article Snippet: NIH 3T3 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% newborn calf serum (NCS) under standard conditions of temperature (37°C), humidity (95°C), and carbon dioxide (5%).

    Techniques: Incubation

    Effect of caspase protease inhibitors on Rac-induced apoptosis. Rac1 and control NIH 3T3 cells were incubated in either DMEM supplemented with 10% NCS (+) or DMEM without serum (−). The caspase-3 inhibitor acetyl-DEVD-CHO (50 μM) (A) or the caspase-1 inhibitor acetyl-YVAD-CHO (100 μM) (B) was added and cells incubated for an additional period of 24 h. After this period, cells were analyzed for induction of apoptosis by the DNA fragmentation analysis as described in MATERIALS AND METHODS. Where indicated, DMEM supplemented with PDGF (100 nM) was used. Control, NIH 3T3 cells; Rac1, NIH 3T3 cells overexpressing the mutated Rac1-QL protein.

    Journal: Molecular Biology of the Cell

    Article Title: Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

    doi:

    Figure Lengend Snippet: Effect of caspase protease inhibitors on Rac-induced apoptosis. Rac1 and control NIH 3T3 cells were incubated in either DMEM supplemented with 10% NCS (+) or DMEM without serum (−). The caspase-3 inhibitor acetyl-DEVD-CHO (50 μM) (A) or the caspase-1 inhibitor acetyl-YVAD-CHO (100 μM) (B) was added and cells incubated for an additional period of 24 h. After this period, cells were analyzed for induction of apoptosis by the DNA fragmentation analysis as described in MATERIALS AND METHODS. Where indicated, DMEM supplemented with PDGF (100 nM) was used. Control, NIH 3T3 cells; Rac1, NIH 3T3 cells overexpressing the mutated Rac1-QL protein.

    Article Snippet: NIH 3T3 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% newborn calf serum (NCS) under standard conditions of temperature (37°C), humidity (95°C), and carbon dioxide (5%).

    Techniques: Incubation

    Caspase-3 activation in apoptosis induced by Rac1. NIH 3T3 cells constitutively expressing the Rac1QL protein and cells transfected with the empty plasmid (c) were cultivated for 24 h in DMEM supplemented with 10% new born calf serum (+) or DMEM without serum (−). The cells were collected at 0, 6, 12, and 24 h after treatment and cytoplasmic extracts were tested for protease activity by using specific substrates as indicated in MATERIALS AND METHODS. Caspase-1 and caspase-3 enzyme activity is expressed as fold activation over basal levels. Data are representative of four experiments performed in triplicates, with similar results. Bars represent SD.

    Journal: Molecular Biology of the Cell

    Article Title: Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

    doi:

    Figure Lengend Snippet: Caspase-3 activation in apoptosis induced by Rac1. NIH 3T3 cells constitutively expressing the Rac1QL protein and cells transfected with the empty plasmid (c) were cultivated for 24 h in DMEM supplemented with 10% new born calf serum (+) or DMEM without serum (−). The cells were collected at 0, 6, 12, and 24 h after treatment and cytoplasmic extracts were tested for protease activity by using specific substrates as indicated in MATERIALS AND METHODS. Caspase-1 and caspase-3 enzyme activity is expressed as fold activation over basal levels. Data are representative of four experiments performed in triplicates, with similar results. Bars represent SD.

    Article Snippet: NIH 3T3 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% newborn calf serum (NCS) under standard conditions of temperature (37°C), humidity (95°C), and carbon dioxide (5%).

    Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Activity Assay

    Rac1-induced apoptosis progress in the absence of cytochrome c accumulation in the cytosol. (Left) NIH 3T3 cells (Control) and Rac1-expressing cells (Rac1) were incubated in DMEM medium with (+) or without (−) serum for 24 h. After that, cells extracts were obtained and processed for cytochrome c analysis. Both cytosolic (Cytosol) and mitochondrial preparations (Mitochond.) were tested for the presence of cytochrome c by Western blot analysis, by using a monoclonal antibody against cytochrome c . Cyt. C indicates purified cytochrome c . The arrow indicates the position of the cytochrome c . (Right) Parallel cultures of NIH 3T3 cells (Control) and Rac1-expressing cells (Rac1) were incubated in DMEM medium with (+) or without (−) serum for 24 h cells and then analyzed for induction of apoptosis by the DNA fragmentation analysis as described in MATERIALS AND METHODS.

    Journal: Molecular Biology of the Cell

    Article Title: Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

    doi:

    Figure Lengend Snippet: Rac1-induced apoptosis progress in the absence of cytochrome c accumulation in the cytosol. (Left) NIH 3T3 cells (Control) and Rac1-expressing cells (Rac1) were incubated in DMEM medium with (+) or without (−) serum for 24 h. After that, cells extracts were obtained and processed for cytochrome c analysis. Both cytosolic (Cytosol) and mitochondrial preparations (Mitochond.) were tested for the presence of cytochrome c by Western blot analysis, by using a monoclonal antibody against cytochrome c . Cyt. C indicates purified cytochrome c . The arrow indicates the position of the cytochrome c . (Right) Parallel cultures of NIH 3T3 cells (Control) and Rac1-expressing cells (Rac1) were incubated in DMEM medium with (+) or without (−) serum for 24 h cells and then analyzed for induction of apoptosis by the DNA fragmentation analysis as described in MATERIALS AND METHODS.

    Article Snippet: NIH 3T3 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% newborn calf serum (NCS) under standard conditions of temperature (37°C), humidity (95°C), and carbon dioxide (5%).

    Techniques: Expressing, Incubation, Western Blot, Purification

    Induction of apoptosis by overexpression of the human Rac protein needs mRNA and proteins synthesis. (A) NIH 3T3 cells were transfected with the appropriate plasmids carrying the human gene rac -1 activated by a Leu 61 mutation (QL mutant). Pools of transfected cells were selected for geneticin resistance and equivalent amounts of protein lysates were analyzed by Western blot for the level of expression of the protein by using a specific antibody. Control indicates extracts from cells transfected with the empty vector, pLNCX. Rac1 indicates the constitutively active pLNCX-Rac1 QL mutant. The arrow on the right indicates the position of Rac1QL protein. (B) Analysis of apoptosis by the DNA fragmentation assay after serum deprivation. Cells were incubated for 24 h in DMEM with or without serum and in presence of 50 μM cycloheximide (CHX), actinomycin D (ACT. D). Gel shows the DNA ladder after staining with ethidium bromide.

    Journal: Molecular Biology of the Cell

    Article Title: Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

    doi:

    Figure Lengend Snippet: Induction of apoptosis by overexpression of the human Rac protein needs mRNA and proteins synthesis. (A) NIH 3T3 cells were transfected with the appropriate plasmids carrying the human gene rac -1 activated by a Leu 61 mutation (QL mutant). Pools of transfected cells were selected for geneticin resistance and equivalent amounts of protein lysates were analyzed by Western blot for the level of expression of the protein by using a specific antibody. Control indicates extracts from cells transfected with the empty vector, pLNCX. Rac1 indicates the constitutively active pLNCX-Rac1 QL mutant. The arrow on the right indicates the position of Rac1QL protein. (B) Analysis of apoptosis by the DNA fragmentation assay after serum deprivation. Cells were incubated for 24 h in DMEM with or without serum and in presence of 50 μM cycloheximide (CHX), actinomycin D (ACT. D). Gel shows the DNA ladder after staining with ethidium bromide.

    Article Snippet: NIH 3T3 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% newborn calf serum (NCS) under standard conditions of temperature (37°C), humidity (95°C), and carbon dioxide (5%).

    Techniques: Over Expression, Transfection, Mutagenesis, Western Blot, Expressing, Plasmid Preparation, DNA Fragmentation Assay, Incubation, Activated Clotting Time Assay, Staining

    Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in N2B27 conditions with selection for geneticin (G418)

    Journal: Nature cell biology

    Article Title: A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages

    doi: 10.1038/ncb2742

    Figure Lengend Snippet: Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites. ( a ) Phase images of PB-Oct4 iPSCs −/− treated or untreated with 4OHT for 24 h and subsequently cultured in N2B27 conditions with selection for geneticin (G418)

    Article Snippet: Epiblast stem cells were cultured in N2B27 medium (DMEM/F12 and Neurobasal (both Gibco) in a 1:1 ratio, 1× penicillin/streptomycin, 0.1 mM 2-mercaptoethanol, 2 mM l -glutamine, 1:200 N2 (StemCells) and 1:100 B27 supplement) supplemented with 12 ng ml−1 Fgf2 and 20 ng ml−1 Activin A (home-made).

    Techniques: Cell Culture, Selection

    Growth and proliferation of esophagogastric junction cells in vitro . (A) Cells obtained by EI in sub-culture. (A-a) Cell morphology of each group after sub-culture to the third generation. Magnification, x200; scale bar, 200 µm. (A-b) Cell proliferation curves in SMCM (third generation; red curve) and DMEM/F12 containing 10% newborn bovine serum (10%-F12; second generation; black curve). n=7. Experiments were perfomed in duplicate. A typical ‘S’ curve was observed in SMCM; in 10%-F12, cell proliferation was largely stopped. (B) Representative cell morphology of EC muscle cells. (B-a) Typical structure of ‘hills and valleys’ for primary (two images above on 10th day after adherence) and sub-cultured (two images below, second generation) cells obtained by EI. Scale bar, 200 µm. Cells cultured in vitro grew in a uniform direction as ‘hills and valleys’, as determined by in topographical mapping. (B-b) An illustration of cell morphology. Magnification, x400; scale bar, 200 µm. Cells were spindle- or long-spindle-shaped, but not uniform; some were rod- or besom-like. Pseudopods of cells differed. (B-c) As the number of passages increased, spindle cells became larger and deformed. Magnification, x200; scale bar, 200 µm. Sizes and morphologies of the fifth (middle) and eighth (right) generation cells were compared with primary spindle cells (left), after 36 h of adherence. EI, enzyme-injected; SMCM, smooth muscle cell medium; Clasp, clasp fiber; Sling, sling fiber; EC, esophageal circular; EL, esophageal longitudinal; GC-S, gastric circular muscle near sling in gastric bottom; GC-C, gastric circular muscle near clasp in lesser gastric curvature; OD, optical density; 0.5-EI-4, 0.5 mg/ml collagenase II solution injected into tissues at 4˚C; 1-EI-4, 1 mg/ml collagenase II solution injected into the tissues at 4˚C; T, Trypsin.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction

    doi: 10.3892/etm.2020.8560

    Figure Lengend Snippet: Growth and proliferation of esophagogastric junction cells in vitro . (A) Cells obtained by EI in sub-culture. (A-a) Cell morphology of each group after sub-culture to the third generation. Magnification, x200; scale bar, 200 µm. (A-b) Cell proliferation curves in SMCM (third generation; red curve) and DMEM/F12 containing 10% newborn bovine serum (10%-F12; second generation; black curve). n=7. Experiments were perfomed in duplicate. A typical ‘S’ curve was observed in SMCM; in 10%-F12, cell proliferation was largely stopped. (B) Representative cell morphology of EC muscle cells. (B-a) Typical structure of ‘hills and valleys’ for primary (two images above on 10th day after adherence) and sub-cultured (two images below, second generation) cells obtained by EI. Scale bar, 200 µm. Cells cultured in vitro grew in a uniform direction as ‘hills and valleys’, as determined by in topographical mapping. (B-b) An illustration of cell morphology. Magnification, x400; scale bar, 200 µm. Cells were spindle- or long-spindle-shaped, but not uniform; some were rod- or besom-like. Pseudopods of cells differed. (B-c) As the number of passages increased, spindle cells became larger and deformed. Magnification, x200; scale bar, 200 µm. Sizes and morphologies of the fifth (middle) and eighth (right) generation cells were compared with primary spindle cells (left), after 36 h of adherence. EI, enzyme-injected; SMCM, smooth muscle cell medium; Clasp, clasp fiber; Sling, sling fiber; EC, esophageal circular; EL, esophageal longitudinal; GC-S, gastric circular muscle near sling in gastric bottom; GC-C, gastric circular muscle near clasp in lesser gastric curvature; OD, optical density; 0.5-EI-4, 0.5 mg/ml collagenase II solution injected into tissues at 4˚C; 1-EI-4, 1 mg/ml collagenase II solution injected into the tissues at 4˚C; T, Trypsin.

    Article Snippet: In the present study, primary cells were cultured in DMEM/F12 containing 10% NBS (10%-F12) and patented SMCM; similar morphological features of EGJ cells were observed in a previous study for VSMCs in vitro ( ).

    Techniques: In Vitro, Cell Culture, Injection

    Efficiency of the VPA Treatment (A) AFS cells transfected with OCT4-GFP or NANOG-GFP reporter genes were cultured on plastic culture dishes in growth medium composed of DMEM supplemented with 10% FBS before being transferred on Matrigel-coated dishes in Nutristem medium for 7 to 14 days prior to exposure to 1 mM VPA for 5 days (VPA_AFS cells). TRA-1-60 + cells were subsequently single-cell sorted into four 96-well plates and cultured for another 28 days in Nutristem (supplemented with a ROCK inhibitor to increase cloning efficiency) on Matrigel. In parallel, the whole VPA_AFS cell population was also maintained in culture for 28 days. (B) The number of OCT4-GFP + or NANOG-GFP + clones was monitored at 7, 14, and 28 days in the 96-well plates, and the GFP intensity was recorded at 7 and 28 days using an optical plate reader. (C) TRA-1-60 expression was assessed by flow cytometry (the red tracing shows the isotype control, and the blue tracing shows the primary antibody) in the VPA_AFS cell population after 28 days in culture in Nutristem. (D) OCT4-GFP was validated using immunosfluorescence in hESCs using OCT4A-specific antibody.

    Journal: Molecular Therapy

    Article Title: Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency

    doi: 10.1016/j.ymthe.2016.11.014

    Figure Lengend Snippet: Efficiency of the VPA Treatment (A) AFS cells transfected with OCT4-GFP or NANOG-GFP reporter genes were cultured on plastic culture dishes in growth medium composed of DMEM supplemented with 10% FBS before being transferred on Matrigel-coated dishes in Nutristem medium for 7 to 14 days prior to exposure to 1 mM VPA for 5 days (VPA_AFS cells). TRA-1-60 + cells were subsequently single-cell sorted into four 96-well plates and cultured for another 28 days in Nutristem (supplemented with a ROCK inhibitor to increase cloning efficiency) on Matrigel. In parallel, the whole VPA_AFS cell population was also maintained in culture for 28 days. (B) The number of OCT4-GFP + or NANOG-GFP + clones was monitored at 7, 14, and 28 days in the 96-well plates, and the GFP intensity was recorded at 7 and 28 days using an optical plate reader. (C) TRA-1-60 expression was assessed by flow cytometry (the red tracing shows the isotype control, and the blue tracing shows the primary antibody) in the VPA_AFS cell population after 28 days in culture in Nutristem. (D) OCT4-GFP was validated using immunosfluorescence in hESCs using OCT4A-specific antibody.

    Article Snippet: Anchorage-independent growth was determined by suspending VPA_AFS cells and NTERA2 cells (5 × 103 cells) in DMEM containing 10% FBS and 0.3% low-melting agarose (GIBCO BRL).

    Techniques: Transfection, Cell Culture, Clone Assay, Expressing, Flow Cytometry, Cytometry