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  • 99
    Thermo Fisher celltrace cfse
    hfcMSCs exhibit immunomodulatory effects on CD4 + lymphocytes through regulated PD-1 expression and interaction. a Histograms showing <t>CellTrace™</t> <t>CFSE</t> dilution in a representative sample of CD4+ T cells following 3 days of incubation in the presence or absence of hfcMSCs and/or blocking αPD-1 antibody. b The effect of hfcMSCs on CD4+ T cell division, shown as proliferation index, after co-culture with hfcMSCs in the presence or absence of blocking αPD-1 antibody. c The presence of hfcMSCs decreased the median fluorescence intensity (MFI) cell surface expression of PD-1 on isolated, activated CD4+CD25+ T cells. The same effect was observed when the cell types were separated by a transwell filter, indicating a predominant role for soluble mediators. d Effects of hfcMSCs on CD4+ T cell activation, depending on cell/cell contact or soluble mediators, shown as IL-2RA (CD25) expression after 3 days of direct co-culture or separation of the two cell types with transwell filters, in the presence or absence of blocking αPD-1 antibody. e The combined bar graph/scatter dot plot depicts T cell proliferation as incorporation of 3 H-thymidine after direct co-culture with hfcMSCs, followed by restimulation with IL-2. The experiments were performed on cells from four different hfcMSC donors ( n = 4). Data from a – d were derived from two T cell donors ( n = 8). Graph data are presented as mean ± SEM. * P
    Celltrace Cfse, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher celltrace
    Defective homing of adoptively transferred WIPΔABD CD4 + T cells to cutaneous sites of Ag challenge in WT recipient mice. (A) Tritiated thymidine ( 3 H-Td) proliferation and IL-2 and IFN-γ production by splenocytes from WIPΔABD and WT mice on the DO11.10 background after stimulation with OVA ( n = 5 for each group). n.d., not detected. (B and C) Representative FACS analysis of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background for surface expression of CCR4, CD43, and PSGL-1 (B) and integrins LFA-1 and VLA-4 (C) ( n = 5 for each group). The solid gray curve in the histogram represents the isotype control. (D) Adhesion of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background to ICAM-1 and VCAM-1 under conditions of physiologic shear stress flow (1 dyn/cm 2 ; n = 2 for each group). (E) Representative FACS analysis of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice. (F) Percentages and numbers of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 5 for each group). (G) Percentages of cells that have proliferated, as determined by dilution of <t>CellTrace</t> Violet (left panel), and of annexin V + cells (right panel) among CD4 + KJ1-26 + cells in OVA-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 4 for each group). Columns and bars represent means ± the SEM. **, P
    Celltrace, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher celltrace ef670
    Defective homing of adoptively transferred WIPΔABD CD4 + T cells to cutaneous sites of Ag challenge in WT recipient mice. (A) Tritiated thymidine ( 3 H-Td) proliferation and IL-2 and IFN-γ production by splenocytes from WIPΔABD and WT mice on the DO11.10 background after stimulation with OVA ( n = 5 for each group). n.d., not detected. (B and C) Representative FACS analysis of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background for surface expression of CCR4, CD43, and PSGL-1 (B) and integrins LFA-1 and VLA-4 (C) ( n = 5 for each group). The solid gray curve in the histogram represents the isotype control. (D) Adhesion of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background to ICAM-1 and VCAM-1 under conditions of physiologic shear stress flow (1 dyn/cm 2 ; n = 2 for each group). (E) Representative FACS analysis of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice. (F) Percentages and numbers of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 5 for each group). (G) Percentages of cells that have proliferated, as determined by dilution of <t>CellTrace</t> Violet (left panel), and of annexin V + cells (right panel) among CD4 + KJ1-26 + cells in OVA-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 4 for each group). Columns and bars represent means ± the SEM. **, P
    Celltrace Ef670, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher celltrace farred
    Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM <t>CellTrace™</t> Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ <t>FarRed</t> (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P
    Celltrace Farred, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher celltrace fr
    Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM <t>CellTrace™</t> Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ <t>FarRed</t> (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P
    Celltrace Fr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher celltrace csfe
    Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM <t>CellTrace™</t> Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ <t>FarRed</t> (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P
    Celltrace Csfe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher celltracer farred
    Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM <t>CellTrace™</t> Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ <t>FarRed</t> (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P
    Celltracer Farred, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher celltrace vioblue
    Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM <t>CellTrace™</t> Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ <t>FarRed</t> (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P
    Celltrace Vioblue, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher celltrace violet
    Short, but not prolonged treatment of salmon MPs with CpGs upregulates their allostimulatory activity. (A) , adherent HK MPs were stained with CFSE upon 24 h of stimulation with 2 μM CpGs. Splenocytes stained with <t>CellTrace</t> Violet (CTV) were used as responders. The dot plots show that a significant number of stimulators take up CTV which necessitates their staining with CFSE in order to distinguish them from proliferating responders with reduced CTV staining. The CFSE-negative events were gated and displayed in histograms to quantify the percentage of proliferating splenocytes with reduced CTV staining. In the representative histogram (bottom right), responders were cultured with non-stimulated (NS) MPs from another individual (filled gray area), and stimulators pretreated with CpGs for 24 h (black contour). (B) , Stimulators pretreated with CpGs for 24 h but not 7 days show increased allostimulatory capacity ( n = 5). Autologous stimulators did not significantly activate splenocyte proliferation even after pretreatment with CpGs for 24 h (* p
    Celltrace Violet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4829 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher celltrace blue kit
    Vδ2-matured DC and B cells stimulate proliferation of resting allogeneic T cells . DC or B cells were co-cultured with HMB-PP-expanded human Vδ2 T cells in the absence or presence of HMB-PP (denoted H). After 24 h <t>CellTrace-labeled</t> allogeneic resting αβ T cells were added at a ratio of 10:1 and cultured for 6 days. (A) Representative dot plot showing proliferating T cells. (B) Histogram showing proliferating T cells (green peaks) versus unstimulated T cells (red peak) by flow cytometric analysis of cell trace dilution. (C) Average (±SEM) percentage of proliferating T cells when cultured with Vδ2-matured DC ( n = 10; left) and levels of IFN-γ and IL-4 secreted by cultures of Vδ2 T cell matured DC with αβ T cells ( n = 6–10). (D) Average (±SEM) percentage of proliferating T cells when cultured with Vδ2-matured B cells ( n = 4) and levels of IFN-γ and IL-4 secreted by cultures of Vδ2 T cell matured DC with αβ T cells ( n = 4). * p
    Celltrace Blue Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher celltrace red
    Effect of decreased ZnT8 expression on MIN6 cell labile Zn content and dispersed islet cell viability. (A) MIN6 cells were analyzed using flow cytometry after staining with the Zn-specific fluorescent probe FluoZin-3 AM and live-cell dye <t>CellTrace</t> calcein
    Celltrace Red, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher celltrace yellow
    Ras-driven invasion requires RalB, rather than MAPK and PI3K. ( A ) Comparison of invasive capability of HEK-HT and HEK-HT-H-RasV12 cells through a thin matrigel layer using Transwell invasion assay (6 hr). Representative images of invading cells stained with DAPI are shown below the graph. Scale bar, 50 µm. Graph shows the mean ±SEM. Each dot represents one experiment. For statistics Student t-test was used. ( B ) Comparison of invasive capability of HEK-HT and HEK-HT-HRasV12 cells through a 3D bovine collagen gel (2.3 mg/ml) using Inverted invasion assay (4 days). Cells were live stained with <t>CellTrace.</t> Invasion was quantified as mean fluorescence of confocal fields acquired every 5 µm starting from below the porous membrane till 200 µm distance inside the gel. Graph shows the mean ±SD of measurements in duplicate from three experiments. Representative images are shown below the graph. Scale bar, 250 µm. Statistical comparison of the curves was done using Student t-test at every 25 µm. ( C ) RalB but not RalA is required for HEK-HTRasV12 invasion through a matrigel layer (Transwell invasion assay). Cells were transfected with the indicated siRNAs. For statistics one-way ANOVA test was used. ( D ) RalB is required for HEK-HTRasV12 invasion through a 3D collagen gel (Inverted invasion assay). Invasion was quantified as in panel B. ( E ) Comparison of invasive capability of HEK-HT-H-RasV12, HEK-HT-H-RasV12G37 and PIK90-treated HEK-HT-H-RasV12G37 cell lines using Transwell invasion assay. The combination of the additional G37 mutation and treatment with the PI3K inhibitor PIK90 (10 µM, treatment started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion) impairs RasV12-dependent MAPK and PI3K hyperactivation, conserving only the activation of Ral pathway. For statistics one-way ANOVA test was used. ( F ) Comparison of invasive capability of HEK-HT-H-RasV12 cells treated with DMSO, with the MEK inhibitor Trametinib (10 nM), with the PI3K inhibitor PIK90 (2.5 μM), or with both Trametinib and PIK90. Treatments were started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion. Cell lysates were prepared at 1 hr post-treatment. On the right, representative western blots for total Akt and phospho-Akt (Ser437), and for total Erk and phospho-Erk (Thr202/Tyr204), show the efficient pharmacological blockage of MAPK and PI3K pathways. Quantifications of Akt and Erk phosphorylation, calculated as p-Akt/total Akt or p-Erk/total Erk ratios, and normalized for HEK-HT-HRasV12 without drugs, are shown below the WBs. For statistics one-way ANOVA test was used. *p
    Celltrace Yellow, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher celltrace calcein violet
    Effect of celastrol concentration on cell viability in cerebral cortical cultures. Cell viability effects were not detected at celastrol concentrations of up to 1.5 μM. Cell viability was assayed by <t>CellTrace</t> <t>calcein</t> ( green ) identifying live cells, MAP2, a marker for neuronal cellular processes ( red ) and dead cell stain ( blue ). Celastrol treatment at 3 μM was included as a positive control that resulted in the retraction of cellular processes ( red ), a decrease in the live cell signal ( green ), and an increase in dead cell signal ( blue ). Scale bar = 20 μM
    Celltrace Calcein Violet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    InvivoGen celltrace cfse
    Effect of celastrol concentration on cell viability in cerebral cortical cultures. Cell viability effects were not detected at celastrol concentrations of up to 1.5 μM. Cell viability was assayed by <t>CellTrace</t> <t>calcein</t> ( green ) identifying live cells, MAP2, a marker for neuronal cellular processes ( red ) and dead cell stain ( blue ). Celastrol treatment at 3 μM was included as a positive control that resulted in the retraction of cellular processes ( red ), a decrease in the live cell signal ( green ), and an increase in dead cell signal ( blue ). Scale bar = 20 μM
    Celltrace Cfse, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific celltrace violet
    Effect of celastrol concentration on cell viability in cerebral cortical cultures. Cell viability effects were not detected at celastrol concentrations of up to 1.5 μM. Cell viability was assayed by <t>CellTrace</t> <t>calcein</t> ( green ) identifying live cells, MAP2, a marker for neuronal cellular processes ( red ) and dead cell stain ( blue ). Celastrol treatment at 3 μM was included as a positive control that resulted in the retraction of cellular processes ( red ), a decrease in the live cell signal ( green ), and an increase in dead cell signal ( blue ). Scale bar = 20 μM
    Celltrace Violet, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher celltracer violet
    High doses of Nec-1 inhibit T cell proliferation. (A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD −/− mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [ 3 H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3 + T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3 + primary T cells were labeled with <t>CellTracer</t> Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3 + T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD −/− RIP1 −/− DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).
    Celltracer Violet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson celltrace violet
    High doses of Nec-1 inhibit T cell proliferation. (A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD −/− mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [ 3 H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3 + T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3 + primary T cells were labeled with <t>CellTracer</t> Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3 + T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD −/− RIP1 −/− DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).
    Celltrace Violet, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher celltrace labeled
    Sustained IA b expression in autophagy-deficient tumor-derived M-MDSCs endows their immunogenic properties. ( A ) Representative flow cytometric analysis of <t>CellTrace-labeled</t> OTII CD4 + T cells cultured with M-MDSCs of Atg5 ΔLysM and control B16-F10–inoculated mice in the presence of OVA peptide, n = 5 mice per group. ( B ) Gating strategy and frequencies of CD25 + (* P = 0.0236) and CD44 + (** P = 0.0116) OTII CD4 + T cells adoptively transferred in Atg5 ΔLysM and control tumor-bearing mice, n = 3 mice per group. ( C ) Relative March1 expression in M-MDSCs following transfection with siRNA for March1 or scramble si (** P = 0.0006, n = 3 mice per group). ( D ) Representative histograms and MFI for IA b expression (* P = 0.0129) in M-MDSCs following transfection with siRNA for March1 or scramble si ( n = 4 mice per group). For E and F , 4 × 10 5 M-MDSCs transfected with scramble si or si-March1 were s.c. coinjected with 3 × 10 5 B16-F10 cells in C57BL/6 mice ( n = 7 mice per group). ( E ) Tumor volume (* P = 0.0044, ** P = 0.017, *** P
    Celltrace Labeled, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    hfcMSCs exhibit immunomodulatory effects on CD4 + lymphocytes through regulated PD-1 expression and interaction. a Histograms showing CellTrace™ CFSE dilution in a representative sample of CD4+ T cells following 3 days of incubation in the presence or absence of hfcMSCs and/or blocking αPD-1 antibody. b The effect of hfcMSCs on CD4+ T cell division, shown as proliferation index, after co-culture with hfcMSCs in the presence or absence of blocking αPD-1 antibody. c The presence of hfcMSCs decreased the median fluorescence intensity (MFI) cell surface expression of PD-1 on isolated, activated CD4+CD25+ T cells. The same effect was observed when the cell types were separated by a transwell filter, indicating a predominant role for soluble mediators. d Effects of hfcMSCs on CD4+ T cell activation, depending on cell/cell contact or soluble mediators, shown as IL-2RA (CD25) expression after 3 days of direct co-culture or separation of the two cell types with transwell filters, in the presence or absence of blocking αPD-1 antibody. e The combined bar graph/scatter dot plot depicts T cell proliferation as incorporation of 3 H-thymidine after direct co-culture with hfcMSCs, followed by restimulation with IL-2. The experiments were performed on cells from four different hfcMSC donors ( n = 4). Data from a – d were derived from two T cell donors ( n = 8). Graph data are presented as mean ± SEM. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Immunomodulatory effects of interferon-γ on human fetal cardiac mesenchymal stromal cells

    doi: 10.1186/s13287-019-1489-1

    Figure Lengend Snippet: hfcMSCs exhibit immunomodulatory effects on CD4 + lymphocytes through regulated PD-1 expression and interaction. a Histograms showing CellTrace™ CFSE dilution in a representative sample of CD4+ T cells following 3 days of incubation in the presence or absence of hfcMSCs and/or blocking αPD-1 antibody. b The effect of hfcMSCs on CD4+ T cell division, shown as proliferation index, after co-culture with hfcMSCs in the presence or absence of blocking αPD-1 antibody. c The presence of hfcMSCs decreased the median fluorescence intensity (MFI) cell surface expression of PD-1 on isolated, activated CD4+CD25+ T cells. The same effect was observed when the cell types were separated by a transwell filter, indicating a predominant role for soluble mediators. d Effects of hfcMSCs on CD4+ T cell activation, depending on cell/cell contact or soluble mediators, shown as IL-2RA (CD25) expression after 3 days of direct co-culture or separation of the two cell types with transwell filters, in the presence or absence of blocking αPD-1 antibody. e The combined bar graph/scatter dot plot depicts T cell proliferation as incorporation of 3 H-thymidine after direct co-culture with hfcMSCs, followed by restimulation with IL-2. The experiments were performed on cells from four different hfcMSC donors ( n = 4). Data from a – d were derived from two T cell donors ( n = 8). Graph data are presented as mean ± SEM. * P

    Article Snippet: Where cell proliferation was assessed, PBMCs were incubated with 0.25 μM CellTrace™ CFSE (ThermoFisher Scientific) for 7 min at 37 °C.

    Techniques: Expressing, Incubation, Blocking Assay, Co-Culture Assay, Fluorescence, Isolation, Activation Assay, Derivative Assay

    Defective homing of adoptively transferred WIPΔABD CD4 + T cells to cutaneous sites of Ag challenge in WT recipient mice. (A) Tritiated thymidine ( 3 H-Td) proliferation and IL-2 and IFN-γ production by splenocytes from WIPΔABD and WT mice on the DO11.10 background after stimulation with OVA ( n = 5 for each group). n.d., not detected. (B and C) Representative FACS analysis of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background for surface expression of CCR4, CD43, and PSGL-1 (B) and integrins LFA-1 and VLA-4 (C) ( n = 5 for each group). The solid gray curve in the histogram represents the isotype control. (D) Adhesion of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background to ICAM-1 and VCAM-1 under conditions of physiologic shear stress flow (1 dyn/cm 2 ; n = 2 for each group). (E) Representative FACS analysis of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice. (F) Percentages and numbers of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 5 for each group). (G) Percentages of cells that have proliferated, as determined by dilution of CellTrace Violet (left panel), and of annexin V + cells (right panel) among CD4 + KJ1-26 + cells in OVA-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 4 for each group). Columns and bars represent means ± the SEM. **, P

    Journal: Molecular and Cellular Biology

    Article Title: Binding of WIP to Actin Is Essential for T Cell Actin Cytoskeleton Integrity and Tissue Homing

    doi: 10.1128/MCB.00533-14

    Figure Lengend Snippet: Defective homing of adoptively transferred WIPΔABD CD4 + T cells to cutaneous sites of Ag challenge in WT recipient mice. (A) Tritiated thymidine ( 3 H-Td) proliferation and IL-2 and IFN-γ production by splenocytes from WIPΔABD and WT mice on the DO11.10 background after stimulation with OVA ( n = 5 for each group). n.d., not detected. (B and C) Representative FACS analysis of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background for surface expression of CCR4, CD43, and PSGL-1 (B) and integrins LFA-1 and VLA-4 (C) ( n = 5 for each group). The solid gray curve in the histogram represents the isotype control. (D) Adhesion of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background to ICAM-1 and VCAM-1 under conditions of physiologic shear stress flow (1 dyn/cm 2 ; n = 2 for each group). (E) Representative FACS analysis of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice. (F) Percentages and numbers of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 5 for each group). (G) Percentages of cells that have proliferated, as determined by dilution of CellTrace Violet (left panel), and of annexin V + cells (right panel) among CD4 + KJ1-26 + cells in OVA-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 4 for each group). Columns and bars represent means ± the SEM. **, P

    Article Snippet: Purified CD4+ cells were stained with CellTrace (Molecular Probes) and 15 × 106 cells were adoptively transferred to naive WT or WIPΔABD BALB/c recipients, which were challenged the same day by i.d. injection in one ear of 20 μl of a 1:1 mixture of 25 μg of OVA323-339 peptide in PBS plus 100 ng of PT-IFA.

    Techniques: Mouse Assay, FACS, Expressing, Flow Cytometry, Derivative Assay

    Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM CellTrace™ Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ FarRed (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P

    Journal: Haematologica

    Article Title: Apoptotic mesenchymal stromal cells induce prostaglandin E2 in monocytes: implications for the monitoring of mesenchymal stromal cell activity

    doi: 10.3324/haematol.2018.214767

    Figure Lengend Snippet: Efferocytosis of apoptotic mesenchymal stromal cells polarizes monocytes into an immunosuppressive phenotype. (A) Apoptotic mesenchymal stromal cells (ApoMSC) were stained with 20 ng/mL pHrodo™ Red-succinimidyl ester (PE) while isolated monocytes were stained with 1 μM CellTrace™ Violet (V450). Monocytes were cultured with different amounts of ApoMSC for 2 h at 37°C, 5% CO2. Lipopolysaccharide (LPS) 100 ng/mL was used to stimulate monocytes. The mean fluorescence intensity (MFI) (PE) within the monocyte population was measured as an indicator of efferocytosis, n=3. (B) MFI (PE) after 2 and 24 h was compared among the different groups, n=3. (C) Confocal images of monocytes efferocytosing ApoMSC after 24 h in culture. The yellow arrows indicate the monocytes engulfing ApoMSC while the white arrows indicate ApoMSC alone. The bar represents 20 μm. (D) Monocytes (2 × 10 5 /well) were co-cultured with ApoMSC (4 × 10 5 /well) for 8 h. CellTrace™ Violet-labeled CD3 T cells (2 × 10 5 /well) were added to the cultures and stimulated by CD3/CD28 beads at a 1:1 cell:bead ratio for 3 days. The percentages of proliferating T cells were measured by flow cytometry according to the loss of fluorescence intensity due to cell division, n=4. (E-G) Intracellular and surface staining was performed to examine the expression of different proteins, including COX2, IDO and PD-L1, in monocytes. The COX2 high , n=3 (E), IDO high , n=4 (F) and PD-L1 high , n=3 (G) populations were gated according to the monocyte alone control (used as a negative control). (H-J) Enzyme-linked immunosorbent assays were performed to assess the amount of PGE2, n=4 (H), IL-10, n=3 (I) and TNF-α, n=6 (J) in cell culture supernatants. (K) To select the monocytes according to efferocytosis, ApoMSC were fluorescent-labeled with CellTrace™ FarRed (APC + ) while monocytes were labeled with CellTrace™ Violet (V450 + ). APC + monocytes were regarded as efferocytosis + and APC − monocytes were regarded as efferocytosis − . (L) Bar charts showing the difference of COX2 high , IDO high and PD-L1 high populations between the efferocytosis + and efferocytosis- monocytes, n=3. Experimental data are expressed as means ± standard deviation. An unpaired t test was used to compare the mean differences between two samples. One-way analysis of variance and the post-hoc Tukey test were used to compare the mean differences among the samples (* P

    Article Snippet: To confirm the association between efferocytosis and the production of these immunosuppressive molecules, ApoMSC were labeled with CellTrace™ FarRed (APC, Thermo Fisher Scientific) ( ).

    Techniques: Staining, Isolation, Cell Culture, Fluorescence, Labeling, Flow Cytometry, Cytometry, Expressing, Negative Control, Standard Deviation

    Short, but not prolonged treatment of salmon MPs with CpGs upregulates their allostimulatory activity. (A) , adherent HK MPs were stained with CFSE upon 24 h of stimulation with 2 μM CpGs. Splenocytes stained with CellTrace Violet (CTV) were used as responders. The dot plots show that a significant number of stimulators take up CTV which necessitates their staining with CFSE in order to distinguish them from proliferating responders with reduced CTV staining. The CFSE-negative events were gated and displayed in histograms to quantify the percentage of proliferating splenocytes with reduced CTV staining. In the representative histogram (bottom right), responders were cultured with non-stimulated (NS) MPs from another individual (filled gray area), and stimulators pretreated with CpGs for 24 h (black contour). (B) , Stimulators pretreated with CpGs for 24 h but not 7 days show increased allostimulatory capacity ( n = 5). Autologous stimulators did not significantly activate splenocyte proliferation even after pretreatment with CpGs for 24 h (* p

    Journal: Frontiers in Immunology

    Article Title: CpGs Induce Differentiation of Atlantic Salmon Mononuclear Phagocytes Into Cells With Dendritic Morphology and a Proinflammatory Transcriptional Profile but an Exhausted Allostimulatory Activity

    doi: 10.3389/fimmu.2019.00378

    Figure Lengend Snippet: Short, but not prolonged treatment of salmon MPs with CpGs upregulates their allostimulatory activity. (A) , adherent HK MPs were stained with CFSE upon 24 h of stimulation with 2 μM CpGs. Splenocytes stained with CellTrace Violet (CTV) were used as responders. The dot plots show that a significant number of stimulators take up CTV which necessitates their staining with CFSE in order to distinguish them from proliferating responders with reduced CTV staining. The CFSE-negative events were gated and displayed in histograms to quantify the percentage of proliferating splenocytes with reduced CTV staining. In the representative histogram (bottom right), responders were cultured with non-stimulated (NS) MPs from another individual (filled gray area), and stimulators pretreated with CpGs for 24 h (black contour). (B) , Stimulators pretreated with CpGs for 24 h but not 7 days show increased allostimulatory capacity ( n = 5). Autologous stimulators did not significantly activate splenocyte proliferation even after pretreatment with CpGs for 24 h (* p

    Article Snippet: CellTrace™ Violet, CellTrace™ CFSE, AlexaFluor546, and Ova-Alexa488, were purchased from Life technologies.

    Techniques: Activity Assay, Staining, Cell Culture

    TCRγδ+LAP+ cells function as APCs and induce Foxp3 in CD4 T cells. ( a ) Soluble ovalbumin (OVA) coupled to Alexa Fluor 488 (OVA-AF488) endocytosis by CD3+TCRγδ+LAP− or CD3+TCRγδ+LAP+ cells after 3 h of culture in vitro at 37 °C ( n =pooled cells from 10 mice per experiment). ( b , c ) Proliferation ( b ) and Foxp3 induction ( c ) in CellTrace Violet-stained naive CD4 T cells from OT-IIxFoxp3-GFP mice co-cultured with OVA 323–339 -loaded CD3+TCRγδ+LAP−, CD3+TCRγδ+LAP+, CD103−CD11c+. or CD103+CD11c+ cells from WT C57BL/6 mice for 4 days at 37 °C ( n =pooled cells from 10 mice per experiment). ( d ) Foxp3 induction in CellTrace Violet-stained naive CD4 T cells from OT-IIxFoxp3-GFP mice co-cultured with OVA 323–339 -loaded TCRγδ+LAP+ cells from WT C57BL/6 mice in the presence or absence of 30 μg ml −1 of anti-LAP mAb for 4 days at 37 °C ( n =pooled cells from 10 mice per experiment). ( e , f ) Proliferation ( e ) and Foxp3 induction ( f ) in CellTrace Violet-stained naive CD4 T cells from OT-IIxFoxp3-GFP mice co-cultured with OVA 323–339 -loaded (or not) CD3+TCRγδ+LAP− or CD3+TCRγδ+LAP+ cells from either WT C57BL/6 or MHC-II−/− mice for 4 days at 37 °C ( n =pooled cells from 10 mice per experiment). These data are representative of at least three independent experiments. One-way analysis of variance followed by Tukey multiple comparisons ( b , e , f ) and Student's t -test ( a , c , d ) were used. ** P

    Journal: Nature Communications

    Article Title: Identification and characterization of latency-associated peptide-expressing γδ T cells

    doi: 10.1038/ncomms9726

    Figure Lengend Snippet: TCRγδ+LAP+ cells function as APCs and induce Foxp3 in CD4 T cells. ( a ) Soluble ovalbumin (OVA) coupled to Alexa Fluor 488 (OVA-AF488) endocytosis by CD3+TCRγδ+LAP− or CD3+TCRγδ+LAP+ cells after 3 h of culture in vitro at 37 °C ( n =pooled cells from 10 mice per experiment). ( b , c ) Proliferation ( b ) and Foxp3 induction ( c ) in CellTrace Violet-stained naive CD4 T cells from OT-IIxFoxp3-GFP mice co-cultured with OVA 323–339 -loaded CD3+TCRγδ+LAP−, CD3+TCRγδ+LAP+, CD103−CD11c+. or CD103+CD11c+ cells from WT C57BL/6 mice for 4 days at 37 °C ( n =pooled cells from 10 mice per experiment). ( d ) Foxp3 induction in CellTrace Violet-stained naive CD4 T cells from OT-IIxFoxp3-GFP mice co-cultured with OVA 323–339 -loaded TCRγδ+LAP+ cells from WT C57BL/6 mice in the presence or absence of 30 μg ml −1 of anti-LAP mAb for 4 days at 37 °C ( n =pooled cells from 10 mice per experiment). ( e , f ) Proliferation ( e ) and Foxp3 induction ( f ) in CellTrace Violet-stained naive CD4 T cells from OT-IIxFoxp3-GFP mice co-cultured with OVA 323–339 -loaded (or not) CD3+TCRγδ+LAP− or CD3+TCRγδ+LAP+ cells from either WT C57BL/6 or MHC-II−/− mice for 4 days at 37 °C ( n =pooled cells from 10 mice per experiment). These data are representative of at least three independent experiments. One-way analysis of variance followed by Tukey multiple comparisons ( b , e , f ) and Student's t -test ( a , c , d ) were used. ** P

    Article Snippet: Next day, the cells were thoroughly washed and incubated at 1:1 ratio with sorted naive (CD4+CD62L+CD44-Foxp3−) cells from OT-IIxFoxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days.

    Techniques: In Vitro, Mouse Assay, Staining, Cell Culture

    Vδ2-matured DC and B cells stimulate proliferation of resting allogeneic T cells . DC or B cells were co-cultured with HMB-PP-expanded human Vδ2 T cells in the absence or presence of HMB-PP (denoted H). After 24 h CellTrace-labeled allogeneic resting αβ T cells were added at a ratio of 10:1 and cultured for 6 days. (A) Representative dot plot showing proliferating T cells. (B) Histogram showing proliferating T cells (green peaks) versus unstimulated T cells (red peak) by flow cytometric analysis of cell trace dilution. (C) Average (±SEM) percentage of proliferating T cells when cultured with Vδ2-matured DC ( n = 10; left) and levels of IFN-γ and IL-4 secreted by cultures of Vδ2 T cell matured DC with αβ T cells ( n = 6–10). (D) Average (±SEM) percentage of proliferating T cells when cultured with Vδ2-matured B cells ( n = 4) and levels of IFN-γ and IL-4 secreted by cultures of Vδ2 T cell matured DC with αβ T cells ( n = 4). * p

    Journal: Frontiers in Immunology

    Article Title: Human Vδ2+ γδ T Cells Differentially Induce Maturation, Cytokine Production, and Alloreactive T Cell Stimulation by Dendritic Cells and B Cells

    doi: 10.3389/fimmu.2014.00650

    Figure Lengend Snippet: Vδ2-matured DC and B cells stimulate proliferation of resting allogeneic T cells . DC or B cells were co-cultured with HMB-PP-expanded human Vδ2 T cells in the absence or presence of HMB-PP (denoted H). After 24 h CellTrace-labeled allogeneic resting αβ T cells were added at a ratio of 10:1 and cultured for 6 days. (A) Representative dot plot showing proliferating T cells. (B) Histogram showing proliferating T cells (green peaks) versus unstimulated T cells (red peak) by flow cytometric analysis of cell trace dilution. (C) Average (±SEM) percentage of proliferating T cells when cultured with Vδ2-matured DC ( n = 10; left) and levels of IFN-γ and IL-4 secreted by cultures of Vδ2 T cell matured DC with αβ T cells ( n = 6–10). (D) Average (±SEM) percentage of proliferating T cells when cultured with Vδ2-matured B cells ( n = 4) and levels of IFN-γ and IL-4 secreted by cultures of Vδ2 T cell matured DC with αβ T cells ( n = 4). * p

    Article Snippet: Alloreactive T cell proliferation Vδ2 T cells were cultured with either B cells or DC in equal numbers in the presence or absence of HMB-PP for 24 h. γδ− PBMC were enriched for CD3+ cells using a magnetic bead cell sorting kit (Miltenyi Biotec) and stained using a CellTrace™ kit (Invitrogen, CA, USA).

    Techniques: Cell Culture, Labeling, Flow Cytometry

    Effect of decreased ZnT8 expression on MIN6 cell labile Zn content and dispersed islet cell viability. (A) MIN6 cells were analyzed using flow cytometry after staining with the Zn-specific fluorescent probe FluoZin-3 AM and live-cell dye CellTrace calcein

    Journal: The Journal of endocrinology

    Article Title: Acute cytokine-mediated downregulation of the zinc transporter ZnT8 alters pancreatic ?-cell function

    doi: 10.1677/JOE-09-0420

    Figure Lengend Snippet: Effect of decreased ZnT8 expression on MIN6 cell labile Zn content and dispersed islet cell viability. (A) MIN6 cells were analyzed using flow cytometry after staining with the Zn-specific fluorescent probe FluoZin-3 AM and live-cell dye CellTrace calcein

    Article Snippet: Cells were subsequently incubated for 1 h in basic media supplemented with 2 mM FluoZin-3 AM (Invitrogen) and 1 mM CellTrace Red (Invitrogen) at 37 °C in 5% CO2 .

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining

    Ras-driven invasion requires RalB, rather than MAPK and PI3K. ( A ) Comparison of invasive capability of HEK-HT and HEK-HT-H-RasV12 cells through a thin matrigel layer using Transwell invasion assay (6 hr). Representative images of invading cells stained with DAPI are shown below the graph. Scale bar, 50 µm. Graph shows the mean ±SEM. Each dot represents one experiment. For statistics Student t-test was used. ( B ) Comparison of invasive capability of HEK-HT and HEK-HT-HRasV12 cells through a 3D bovine collagen gel (2.3 mg/ml) using Inverted invasion assay (4 days). Cells were live stained with CellTrace. Invasion was quantified as mean fluorescence of confocal fields acquired every 5 µm starting from below the porous membrane till 200 µm distance inside the gel. Graph shows the mean ±SD of measurements in duplicate from three experiments. Representative images are shown below the graph. Scale bar, 250 µm. Statistical comparison of the curves was done using Student t-test at every 25 µm. ( C ) RalB but not RalA is required for HEK-HTRasV12 invasion through a matrigel layer (Transwell invasion assay). Cells were transfected with the indicated siRNAs. For statistics one-way ANOVA test was used. ( D ) RalB is required for HEK-HTRasV12 invasion through a 3D collagen gel (Inverted invasion assay). Invasion was quantified as in panel B. ( E ) Comparison of invasive capability of HEK-HT-H-RasV12, HEK-HT-H-RasV12G37 and PIK90-treated HEK-HT-H-RasV12G37 cell lines using Transwell invasion assay. The combination of the additional G37 mutation and treatment with the PI3K inhibitor PIK90 (10 µM, treatment started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion) impairs RasV12-dependent MAPK and PI3K hyperactivation, conserving only the activation of Ral pathway. For statistics one-way ANOVA test was used. ( F ) Comparison of invasive capability of HEK-HT-H-RasV12 cells treated with DMSO, with the MEK inhibitor Trametinib (10 nM), with the PI3K inhibitor PIK90 (2.5 μM), or with both Trametinib and PIK90. Treatments were started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion. Cell lysates were prepared at 1 hr post-treatment. On the right, representative western blots for total Akt and phospho-Akt (Ser437), and for total Erk and phospho-Erk (Thr202/Tyr204), show the efficient pharmacological blockage of MAPK and PI3K pathways. Quantifications of Akt and Erk phosphorylation, calculated as p-Akt/total Akt or p-Erk/total Erk ratios, and normalized for HEK-HT-HRasV12 without drugs, are shown below the WBs. For statistics one-way ANOVA test was used. *p

    Journal: eLife

    Article Title: RalB directly triggers invasion downstream Ras by mobilizing the Wave complex

    doi: 10.7554/eLife.40474

    Figure Lengend Snippet: Ras-driven invasion requires RalB, rather than MAPK and PI3K. ( A ) Comparison of invasive capability of HEK-HT and HEK-HT-H-RasV12 cells through a thin matrigel layer using Transwell invasion assay (6 hr). Representative images of invading cells stained with DAPI are shown below the graph. Scale bar, 50 µm. Graph shows the mean ±SEM. Each dot represents one experiment. For statistics Student t-test was used. ( B ) Comparison of invasive capability of HEK-HT and HEK-HT-HRasV12 cells through a 3D bovine collagen gel (2.3 mg/ml) using Inverted invasion assay (4 days). Cells were live stained with CellTrace. Invasion was quantified as mean fluorescence of confocal fields acquired every 5 µm starting from below the porous membrane till 200 µm distance inside the gel. Graph shows the mean ±SD of measurements in duplicate from three experiments. Representative images are shown below the graph. Scale bar, 250 µm. Statistical comparison of the curves was done using Student t-test at every 25 µm. ( C ) RalB but not RalA is required for HEK-HTRasV12 invasion through a matrigel layer (Transwell invasion assay). Cells were transfected with the indicated siRNAs. For statistics one-way ANOVA test was used. ( D ) RalB is required for HEK-HTRasV12 invasion through a 3D collagen gel (Inverted invasion assay). Invasion was quantified as in panel B. ( E ) Comparison of invasive capability of HEK-HT-H-RasV12, HEK-HT-H-RasV12G37 and PIK90-treated HEK-HT-H-RasV12G37 cell lines using Transwell invasion assay. The combination of the additional G37 mutation and treatment with the PI3K inhibitor PIK90 (10 µM, treatment started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion) impairs RasV12-dependent MAPK and PI3K hyperactivation, conserving only the activation of Ral pathway. For statistics one-way ANOVA test was used. ( F ) Comparison of invasive capability of HEK-HT-H-RasV12 cells treated with DMSO, with the MEK inhibitor Trametinib (10 nM), with the PI3K inhibitor PIK90 (2.5 μM), or with both Trametinib and PIK90. Treatments were started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion. Cell lysates were prepared at 1 hr post-treatment. On the right, representative western blots for total Akt and phospho-Akt (Ser437), and for total Erk and phospho-Erk (Thr202/Tyr204), show the efficient pharmacological blockage of MAPK and PI3K pathways. Quantifications of Akt and Erk phosphorylation, calculated as p-Akt/total Akt or p-Erk/total Erk ratios, and normalized for HEK-HT-HRasV12 without drugs, are shown below the WBs. For statistics one-way ANOVA test was used. *p

    Article Snippet: Cells were live stained with CellTrace yellow (Thermofisher) for 1 hr at 37°C and rapidly imaged by confocal microscopy (Zeiss LSM880, 10x objective, excitation at 545 nm and emission at 605 nm).

    Techniques: Transwell Invasion Assay, Staining, Invasion Assay, Fluorescence, Transfection, Mutagenesis, Activation Assay, Western Blot

    Effect of celastrol concentration on cell viability in cerebral cortical cultures. Cell viability effects were not detected at celastrol concentrations of up to 1.5 μM. Cell viability was assayed by CellTrace calcein ( green ) identifying live cells, MAP2, a marker for neuronal cellular processes ( red ) and dead cell stain ( blue ). Celastrol treatment at 3 μM was included as a positive control that resulted in the retraction of cellular processes ( red ), a decrease in the live cell signal ( green ), and an increase in dead cell signal ( blue ). Scale bar = 20 μM

    Journal: Cell Stress & Chaperones

    Article Title: Localization of heat shock proteins in cerebral cortical cultures following induction by celastrol

    doi: 10.1007/s12192-014-0508-5

    Figure Lengend Snippet: Effect of celastrol concentration on cell viability in cerebral cortical cultures. Cell viability effects were not detected at celastrol concentrations of up to 1.5 μM. Cell viability was assayed by CellTrace calcein ( green ) identifying live cells, MAP2, a marker for neuronal cellular processes ( red ) and dead cell stain ( blue ). Celastrol treatment at 3 μM was included as a positive control that resulted in the retraction of cellular processes ( red ), a decrease in the live cell signal ( green ), and an increase in dead cell signal ( blue ). Scale bar = 20 μM

    Article Snippet: CellTrace calcein violet AM (Invitrogen, Burlington, ON, Canada) was used for identifying live cells.

    Techniques: Concentration Assay, Marker, Staining, Positive Control

    High doses of Nec-1 inhibit T cell proliferation. (A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD −/− mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [ 3 H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3 + T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3 + primary T cells were labeled with CellTracer Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3 + T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD −/− RIP1 −/− DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).

    Journal: PLoS ONE

    Article Title: RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

    doi: 10.1371/journal.pone.0023209

    Figure Lengend Snippet: High doses of Nec-1 inhibit T cell proliferation. (A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD −/− mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [ 3 H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3 + T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3 + primary T cells were labeled with CellTracer Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3 + T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD −/− RIP1 −/− DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).

    Article Snippet: For CellTracer Violet dilution assay, cells were loaded with 5 µM CellTracer Violet as per manufacturer's protocol (Invitrogen).

    Techniques: Purification, Mouse Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Labeling

    Sustained IA b expression in autophagy-deficient tumor-derived M-MDSCs endows their immunogenic properties. ( A ) Representative flow cytometric analysis of CellTrace-labeled OTII CD4 + T cells cultured with M-MDSCs of Atg5 ΔLysM and control B16-F10–inoculated mice in the presence of OVA peptide, n = 5 mice per group. ( B ) Gating strategy and frequencies of CD25 + (* P = 0.0236) and CD44 + (** P = 0.0116) OTII CD4 + T cells adoptively transferred in Atg5 ΔLysM and control tumor-bearing mice, n = 3 mice per group. ( C ) Relative March1 expression in M-MDSCs following transfection with siRNA for March1 or scramble si (** P = 0.0006, n = 3 mice per group). ( D ) Representative histograms and MFI for IA b expression (* P = 0.0129) in M-MDSCs following transfection with siRNA for March1 or scramble si ( n = 4 mice per group). For E and F , 4 × 10 5 M-MDSCs transfected with scramble si or si-March1 were s.c. coinjected with 3 × 10 5 B16-F10 cells in C57BL/6 mice ( n = 7 mice per group). ( E ) Tumor volume (* P = 0.0044, ** P = 0.017, *** P

    Journal: The Journal of Clinical Investigation

    Article Title: Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells

    doi: 10.1172/JCI120888

    Figure Lengend Snippet: Sustained IA b expression in autophagy-deficient tumor-derived M-MDSCs endows their immunogenic properties. ( A ) Representative flow cytometric analysis of CellTrace-labeled OTII CD4 + T cells cultured with M-MDSCs of Atg5 ΔLysM and control B16-F10–inoculated mice in the presence of OVA peptide, n = 5 mice per group. ( B ) Gating strategy and frequencies of CD25 + (* P = 0.0236) and CD44 + (** P = 0.0116) OTII CD4 + T cells adoptively transferred in Atg5 ΔLysM and control tumor-bearing mice, n = 3 mice per group. ( C ) Relative March1 expression in M-MDSCs following transfection with siRNA for March1 or scramble si (** P = 0.0006, n = 3 mice per group). ( D ) Representative histograms and MFI for IA b expression (* P = 0.0129) in M-MDSCs following transfection with siRNA for March1 or scramble si ( n = 4 mice per group). For E and F , 4 × 10 5 M-MDSCs transfected with scramble si or si-March1 were s.c. coinjected with 3 × 10 5 B16-F10 cells in C57BL/6 mice ( n = 7 mice per group). ( E ) Tumor volume (* P = 0.0044, ** P = 0.017, *** P

    Article Snippet: For in vitro suppression assays, highly purified M-MDSCs were sorted from tumors of Atg5ΔLysM and Atg5fl/fl mice and cultured in 96-well round-bottom plates with 1.5 × 105 whole CellTrace-labeled (10 μm, Invitrogen) LN cells (LNCs) of naive C57BL/6 mice in a 1:2 ratio, in the presence of Dynabeads mouse T-activator CD3/CD8 (Life Technologies).

    Techniques: IA, Expressing, Derivative Assay, Flow Cytometry, Labeling, Cell Culture, Mouse Assay, Transfection