Structured Review

Biochrom fbs
HE staining of primary porcine oviduct epithelial cells (POEC) cultured in media M1 ( a ), M2 ( b ), M3 ( c ) and M4 ( d ), respectively, for 6w. M1: Ham’s F12 + 10 % charcoal-stripped <t>FBS;</t> M2: M1 enriched with corresponding <t>3T3-conditioned</t> medium at a ratio of 2:1(V: V); M3: Ham’s F12 + 10 % FBS; M4: M3 enriched with corresponding 3T3-conditioned medium at a ratio of 2:1(V: V); magnification ×400, scale bars 20 μm
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Images

1) Product Images from "Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports"

Article Title: Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports

Journal: Histochemistry and Cell Biology

doi: 10.1007/s00418-015-1351-1

HE staining of primary porcine oviduct epithelial cells (POEC) cultured in media M1 ( a ), M2 ( b ), M3 ( c ) and M4 ( d ), respectively, for 6w. M1: Ham’s F12 + 10 % charcoal-stripped FBS; M2: M1 enriched with corresponding 3T3-conditioned medium at a ratio of 2:1(V: V); M3: Ham’s F12 + 10 % FBS; M4: M3 enriched with corresponding 3T3-conditioned medium at a ratio of 2:1(V: V); magnification ×400, scale bars 20 μm
Figure Legend Snippet: HE staining of primary porcine oviduct epithelial cells (POEC) cultured in media M1 ( a ), M2 ( b ), M3 ( c ) and M4 ( d ), respectively, for 6w. M1: Ham’s F12 + 10 % charcoal-stripped FBS; M2: M1 enriched with corresponding 3T3-conditioned medium at a ratio of 2:1(V: V); M3: Ham’s F12 + 10 % FBS; M4: M3 enriched with corresponding 3T3-conditioned medium at a ratio of 2:1(V: V); magnification ×400, scale bars 20 μm

Techniques Used: Staining, Cell Culture

2) Product Images from "Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression"

Article Title: Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156268

The expression of MMP1. (A) Expression of MMP-1 in 3D mono- and co-culture lung cancer models at 48 h detected by ELISA. The expression of MMP1 in HCC MRC-5 macrophage co-culture group was higher than that in HCC MRC-5 co-culture group, or MRC-5/HCC/macrophage mono-culture groups. There was almost no expression of MMP1 in the HCC/macrophage mono-culture group. (B) Expression of MMP-1 in 3D mono- and co-culture lung cancer model at 48 h detected by Western blotting. In Fig 1B, a, the molecular weight of MMP-1 is 52 kD. From the left to right, the lanes are: HCC mono-culture group (2 x 10 5 cells); MRC-5 mono-culture group (2 x 10 5 cells); MRC-5 and HCC co-culture group (2 x10 5 cells); HCC mono-culture group (1 x 10 6 cells); MRC-5 and HCC co-culture group (1 x 10 6 cells); MRC-5 mono-culture group (1 x 10 6 cells). Expression of MMP-1 in co-culture groups was higher than in mono-culture groups (both 2 x 10 5 cells and 1 x 10 6 cells). Expression of MMP-1 in the 1 x 10 6 cell group was higher than the 2 x 10 5 cell group, regardless of mono-culture or co-culture group designations. In Fig 1B, b, the mean IOD values of the Western blot are shown. (C) Expression of MMP-1 under different co-culture conditions. Expression of MMP1 under 10% FBS and O 2 (10% FBS cell culture medium with O 2 ) was higher than that under w/o FBS and w/o O 2 (without FBS and without O 2 ) at 7 different time points. Furthermore, the expression trend of MMP1 under the condition of w/o FBS and w/o O 2 continued to decline from 120 h.
Figure Legend Snippet: The expression of MMP1. (A) Expression of MMP-1 in 3D mono- and co-culture lung cancer models at 48 h detected by ELISA. The expression of MMP1 in HCC MRC-5 macrophage co-culture group was higher than that in HCC MRC-5 co-culture group, or MRC-5/HCC/macrophage mono-culture groups. There was almost no expression of MMP1 in the HCC/macrophage mono-culture group. (B) Expression of MMP-1 in 3D mono- and co-culture lung cancer model at 48 h detected by Western blotting. In Fig 1B, a, the molecular weight of MMP-1 is 52 kD. From the left to right, the lanes are: HCC mono-culture group (2 x 10 5 cells); MRC-5 mono-culture group (2 x 10 5 cells); MRC-5 and HCC co-culture group (2 x10 5 cells); HCC mono-culture group (1 x 10 6 cells); MRC-5 and HCC co-culture group (1 x 10 6 cells); MRC-5 mono-culture group (1 x 10 6 cells). Expression of MMP-1 in co-culture groups was higher than in mono-culture groups (both 2 x 10 5 cells and 1 x 10 6 cells). Expression of MMP-1 in the 1 x 10 6 cell group was higher than the 2 x 10 5 cell group, regardless of mono-culture or co-culture group designations. In Fig 1B, b, the mean IOD values of the Western blot are shown. (C) Expression of MMP-1 under different co-culture conditions. Expression of MMP1 under 10% FBS and O 2 (10% FBS cell culture medium with O 2 ) was higher than that under w/o FBS and w/o O 2 (without FBS and without O 2 ) at 7 different time points. Furthermore, the expression trend of MMP1 under the condition of w/o FBS and w/o O 2 continued to decline from 120 h.

Techniques Used: Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Molecular Weight, Cell Culture

The expression of VEGF. (A) Expression of VEGF in HCC, MRC-5, and macrophage mono-cultures groups. Expression of VEGF in the HCC mono-culture group was significantly higher than expression in the MRC-5/macrophage mono-culture group under 10% FBS and O 2 culture conditions. (B) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups compared with the HCC mono-culture group. Expression of VEGF in the HCC MRC-5 Macrophage co-culture group was higher than in the HCC MRC-5 co-culture group and the HCC mono-culture group cultured with 10% FBS and O 2 for 48 h. (C) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups under different co-culture conditions. The expression of VEGF in cells cultured w/o FBS (starved of FBS but with O 2 ), w/o FBS and w/o O 2 (without FBS and without O 2 ) was higher than that in 10% FBS or O 2 (10% FBS cell culture medium with O 2 ), while the expression of VEGF in the three different conditions first increased and then decreased.
Figure Legend Snippet: The expression of VEGF. (A) Expression of VEGF in HCC, MRC-5, and macrophage mono-cultures groups. Expression of VEGF in the HCC mono-culture group was significantly higher than expression in the MRC-5/macrophage mono-culture group under 10% FBS and O 2 culture conditions. (B) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups compared with the HCC mono-culture group. Expression of VEGF in the HCC MRC-5 Macrophage co-culture group was higher than in the HCC MRC-5 co-culture group and the HCC mono-culture group cultured with 10% FBS and O 2 for 48 h. (C) Expression of VEGF in HCC, MRC-5, and macrophage co-culture groups under different co-culture conditions. The expression of VEGF in cells cultured w/o FBS (starved of FBS but with O 2 ), w/o FBS and w/o O 2 (without FBS and without O 2 ) was higher than that in 10% FBS or O 2 (10% FBS cell culture medium with O 2 ), while the expression of VEGF in the three different conditions first increased and then decreased.

Techniques Used: Expressing, Co-Culture Assay, Cell Culture

3) Product Images from "Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis"

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

Journal: Cancers

doi: 10.3390/cancers12082085

In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
Figure Legend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Techniques Used: In Vitro, Purification, Cell Culture, Flow Cytometry

Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
Figure Legend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Techniques Used: Purification, Cell Culture, Staining, Flow Cytometry

Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p
Figure Legend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

Techniques Used: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

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Blocking Assay:

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Binding Assay:

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    Biochrom fbs
    BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free <t>RPMI+</t> dialyzed <t>FBS</t> in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.
    Fbs, supplied by Biochrom, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free RPMI+ dialyzed FBS in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.

    Journal: Life Science Alliance

    Article Title: Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes

    doi: 10.26508/lsa.202000700

    Figure Lengend Snippet: BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free RPMI+ dialyzed FBS in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.

    Article Snippet: 0.6 × 106 or 1 × 106 cells were incubated in 1 ml RPMI (Thermo Fisher Scientific) + 1% FBS (Biochrom AG) + 15 μl Indo-1 solution at 37°C for 45 min.

    Techniques: Staining, Incubation, Cell Culture

    In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Journal: Cancers

    Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

    doi: 10.3390/cancers12082085

    Figure Lengend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Article Snippet: In addition, pDCs and T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), titrated to pH ≈ 7.4, 7.0, 6.5, 6.0, and 5.5 using HCl.

    Techniques: In Vitro, Purification, Cell Culture, Flow Cytometry

    Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Journal: Cancers

    Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

    doi: 10.3390/cancers12082085

    Figure Lengend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Article Snippet: In addition, pDCs and T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), titrated to pH ≈ 7.4, 7.0, 6.5, 6.0, and 5.5 using HCl.

    Techniques: Purification, Cell Culture, Staining, Flow Cytometry

    Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

    Journal: Cancers

    Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

    doi: 10.3390/cancers12082085

    Figure Lengend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

    Article Snippet: In addition, pDCs and T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), titrated to pH ≈ 7.4, 7.0, 6.5, 6.0, and 5.5 using HCl.

    Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

    Amino acid starvation causes TSC2 lysosomal localization in a Rag-dependent manner (A,C) TSC2 accumulates on lysosomes upon amino acid deprivation. Confocal micrograph of HEK293FT cells (A) or MEFs (C) treated +/− all amino acids in the presence of dialyzed FBS for the indicated times, stained for TSC2 (green) and the lysosomal marker LAMP2 (red). LAMP2 aggregates do not show TSC2 accumulation in the +aa condition. (B) TSC2 quickly relocalizes away from lysosomes upon amino acid readdition. HEK293FT cells, starved for amino acids in the presence of dialyzed serum for 4h, then re-supplied with amino acid-containing media for 5 minutes. Cells stained and imaged as in (A).

    Journal: Cell

    Article Title: Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

    doi: 10.1016/j.cell.2014.01.024

    Figure Lengend Snippet: Amino acid starvation causes TSC2 lysosomal localization in a Rag-dependent manner (A,C) TSC2 accumulates on lysosomes upon amino acid deprivation. Confocal micrograph of HEK293FT cells (A) or MEFs (C) treated +/− all amino acids in the presence of dialyzed FBS for the indicated times, stained for TSC2 (green) and the lysosomal marker LAMP2 (red). LAMP2 aggregates do not show TSC2 accumulation in the +aa condition. (B) TSC2 quickly relocalizes away from lysosomes upon amino acid readdition. HEK293FT cells, starved for amino acids in the presence of dialyzed serum for 4h, then re-supplied with amino acid-containing media for 5 minutes. Cells stained and imaged as in (A).

    Article Snippet: HEK293FT cells (Invitrogen) and mouse embryonic fibroblasts were cultured in high glucose DMEM, supplemented with 10% FBS (Biochrom), except PTEN−/− MEFS which were also supplemented with 2mM Glutamine.

    Techniques: Staining, Marker

    Lysosomal recruitment of TSC2 depends on the Rag proteins (A-A’) Lysosomal localization of TSC2 upon amino acid removal requires the LAMTOR complex. Confocal micrograph of p14/LAMTOR2 knock-out MEFs (A), or control p14 knock-out MEFs reconstituted to express an EGFP-p14 fusion (A’), treated +/− amino acids for 1h in the presence of dialyzed FBS. Lysosomes marked either with anti-LAMP2 antibody or with the lysosomaly localized EGFP-p14. (B) Lysosomal localization of TSC2 upon amino acid removal requires the Rag proteins. MEFs transfected with control siRNA against Renilla luciferase or siRNAs targeting the various Rag proteins, treated +/− amino acids for 1h in the presence of dialyzed FBS. Cells stained and imaged as in (A). (C) Lysosomal localization of TSC2 upon amino acid removal requires that the Rag proteins change to the inactive conformation. MEFs transfected with control siRNA or siRNAs targeting components of the GATOR1 complex DEPDC5, NPRL2, or NPRL3 required for the Rag proteins to become inactive upon amino acid removal. Cells stained and imaged as in (A).

    Journal: Cell

    Article Title: Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

    doi: 10.1016/j.cell.2014.01.024

    Figure Lengend Snippet: Lysosomal recruitment of TSC2 depends on the Rag proteins (A-A’) Lysosomal localization of TSC2 upon amino acid removal requires the LAMTOR complex. Confocal micrograph of p14/LAMTOR2 knock-out MEFs (A), or control p14 knock-out MEFs reconstituted to express an EGFP-p14 fusion (A’), treated +/− amino acids for 1h in the presence of dialyzed FBS. Lysosomes marked either with anti-LAMP2 antibody or with the lysosomaly localized EGFP-p14. (B) Lysosomal localization of TSC2 upon amino acid removal requires the Rag proteins. MEFs transfected with control siRNA against Renilla luciferase or siRNAs targeting the various Rag proteins, treated +/− amino acids for 1h in the presence of dialyzed FBS. Cells stained and imaged as in (A). (C) Lysosomal localization of TSC2 upon amino acid removal requires that the Rag proteins change to the inactive conformation. MEFs transfected with control siRNA or siRNAs targeting components of the GATOR1 complex DEPDC5, NPRL2, or NPRL3 required for the Rag proteins to become inactive upon amino acid removal. Cells stained and imaged as in (A).

    Article Snippet: HEK293FT cells (Invitrogen) and mouse embryonic fibroblasts were cultured in high glucose DMEM, supplemented with 10% FBS (Biochrom), except PTEN−/− MEFS which were also supplemented with 2mM Glutamine.

    Techniques: Knock-Out, Transfection, Luciferase, Staining

    TSC2 is required for mTOR to localize away from lysosomes upon amino acid removal in a Rheb dependent manner (A-B) mTOR is released from lysosomes upon amino acid removal in control (A) but not TSC2-null MEFs (A’). This is rescued by re-expressing TSC2 + EGFP (to mark transfected cells) but not EGFP alone in the TSC-null MEFs (B). Note that the cell expressing TSC2 and GFP no longer has mTOR accumulated on lysosomes, whereas the surrounding, non-transfected cells retain lysosomally localized mTOR. (C) Defective release of mTOR from lysosomes upon amino acid withdrawal in TSC2-null MEFs is rescued by knocking down Rheb. TSC2-null MEFs transfected with either Rheb siRNAs (upper panels) or control siRNAs (lower panels) treated 1h with +/− amino acid containing medium in the presence of dialyzed FBS.

    Journal: Cell

    Article Title: Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

    doi: 10.1016/j.cell.2014.01.024

    Figure Lengend Snippet: TSC2 is required for mTOR to localize away from lysosomes upon amino acid removal in a Rheb dependent manner (A-B) mTOR is released from lysosomes upon amino acid removal in control (A) but not TSC2-null MEFs (A’). This is rescued by re-expressing TSC2 + EGFP (to mark transfected cells) but not EGFP alone in the TSC-null MEFs (B). Note that the cell expressing TSC2 and GFP no longer has mTOR accumulated on lysosomes, whereas the surrounding, non-transfected cells retain lysosomally localized mTOR. (C) Defective release of mTOR from lysosomes upon amino acid withdrawal in TSC2-null MEFs is rescued by knocking down Rheb. TSC2-null MEFs transfected with either Rheb siRNAs (upper panels) or control siRNAs (lower panels) treated 1h with +/− amino acid containing medium in the presence of dialyzed FBS.

    Article Snippet: HEK293FT cells (Invitrogen) and mouse embryonic fibroblasts were cultured in high glucose DMEM, supplemented with 10% FBS (Biochrom), except PTEN−/− MEFS which were also supplemented with 2mM Glutamine.

    Techniques: Expressing, Transfection