stem cell easysep kit for cd4 + t cells  (STEMCELL Technologies Inc)

Journal: Biosensors

Article Title: A Bioelectronic System to Measure the Glycolytic Metabolism of Activated CD4+ T Cells

doi: 10.3390/bios9010010

Figure Lengend Snippet: Inhibitors of PI3K (LY294002) and mTORC1 (temsirolimus) suppress the acidification and conductivity of culture media of the activated CD4+ T cells. Different pH buffers were initially used to evaluate the performance of the system. ( A ) Changes in the delta mV of different pH reference buffers over a period of 30 min. ( B ) The biosensor response to different concentrations of lactic acid. ( C ) CD4+ T cells were purified by negative selection from HIV-healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/mL), ionomycin (1 ug/mL), and IL-2 (5 ng/mL) for 48 h, replaced with fresh media, and were left untreated (UT) (0.1% carrier DMSO), or treated with either LY294002 (15 uM), temsirolimus (100 nM), or SB201290 (10 uM, p38 inhibitor) for an additional 48 h. Glycolytic activity in the culture medium was measured using the Metabolite Biosensor Device. Cells were cultured at a concentration of 1 × 10 6 cells/mL in supplemented RPMI-1640 medium. The paired T-test was used to determine the significant differences between the treatments.

Article Snippet: The CD4+ T cells were purified from thawed PBMCs from healthy donors using the Human EasySep CD4+ T cell enrichment kit (Stem Cell, Technology Inc, Vancouver, BC, Canada).

Techniques: Purification, Selection, Activation Assay, Activity Assay, Cell Culture, Concentration Assay

Journal: Biosensors

Article Title: A Bioelectronic System to Measure the Glycolytic Metabolism of Activated CD4+ T Cells

doi: 10.3390/bios9010010

Figure Lengend Snippet: Inhibitors of PI3K (LY294002) and mTORC1 (temsirolimus) suppress glucose uptake and lactate production by activated CD4+ T cells. CD4+ T cells were purified by negative selection from HIV-healthy donors, stimulated (as described in the legend of ) with PMA, ionomycin, and IL-2 (5 ng/mL) for 48 h, replaced with fresh media, and then left untreated (UT) or treated with either LY294002 (15 uM), temsirolimus (100 nM), or SB201290 (10 uM, p38 inhibitor). ( A ) Viability of the CD4+ T cells as measured by trypan blue exclusion assay. ( B ) Data showing the amount of glucose taken up by the CD4+ T cells in culture using the GlucMeter. ( C ) 2-NBDG uptake by the CD4+ T cells as measured using flow cytometry. ( D ) L-lactate levels in CD4+ T cell culture medium, measured using an L-lactate assay kit as described in the . All experiments were conducted in duplicates, with three independent experiments. The paired T-test was used to determine significant treatments between the treatments.

Article Snippet: The CD4+ T cells were purified from thawed PBMCs from healthy donors using the Human EasySep CD4+ T cell enrichment kit (Stem Cell, Technology Inc, Vancouver, BC, Canada).

Techniques: Purification, Selection, Trypan Blue Exclusion Assay, Flow Cytometry, Cell Culture, Lactate Assay

Journal: Nature Communications

Article Title: Armc5 deletion causes developmental defects and compromises T-cell immune responses

doi: 10.1038/ncomms13834

Figure Lengend Snippet: ( a ) Armc5 expression in adult mouse using whole-body sections. Upper panel: H/E staining; middle and bottom panels: dark field X-ray film autography with anti-sense (AS) cRNA or sense (S) cRNA as probes, respectively. Bar=1 cm. AG: adrenal gland; B: bone; BM: bone marrow; Cb: cerebellum; K: kidney; Lint: large intestine; LT: lymphatic tissue; Sk: skin; ST: stomach; Th: thymus; VB: vertebrae. ( b ) Armc5 expression in the adult thymus. Upper row: dark field X-ray film autography; lower row: bright field emulsion autoradiography; left column: anti-sense probe; right column: sense probe. Bars=2 mm and 20 μm. Cx: cortex; Me: medulla. ( c ) Armc5 expression in the adult spleen. Upper and middle panels: dark field X-ray film autography, with anti-sense and sense probes, respectively; bottom panel: bright field emulsion autoradiography. Bars=2 mm and 20 μm. WP: white pulp; RP: red pulp; CAr: central artery; Cp: capillary. ( d ) Armc5 mRNA in mouse spleen CD4 and CD8 cells measured by RT-qPCR. Experiments were performed three times. The results of representative experiments are shown. To facilitate comparison, normalized ratios of Armc5 versus β-actin signals (means±s.e.m.) are presented; the 0-h signal ratio of each experiment is considered as 1. ( e ) ARMC5 subcellular localization in L cells was detected immunofluorescence. L cells were transfected with HA-tagged mouse ARMC5-expressing construct or an empty vector, as indicated. ( f ) Phase contract micrographs of views in ( e ). The experiments were conducted three times, and micrographs of a representative experiment are shown. Scale bar: 5 μm.

Article Snippet: Naive CD4 + T cells (CD4 + CD62L + CD44 low ) were isolated from KO or WT mouse Spleen with EasySep mouse naive CD4 + T-cell isolation kits (19765, Stem Cell Technology).

Techniques: Expressing, Staining, Autoradiography, Quantitative RT-PCR, Immunofluorescence, Transfection, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: Armc5 deletion causes developmental defects and compromises T-cell immune responses

doi: 10.1038/ncomms13834

Figure Lengend Snippet: ( a ) Proliferation of spleen CD4 + and CD8 + T cells and B220 + B cells from WT and KO mice according to CFSE staining. CFSE intensity was ascertained by flow cytometry. Experiments were conducted independently 4–6 times. Representative histograms are shown. ( b ) Cell cycle progression of spleen T cells from WT and KO mice. The percentages of cells in G 1 , S and G 2 phases are indicated. Experiments were conducted independently three times. Representative histograms are shown. ( c ) Apoptosis of WT and KO spleen T cells (gated on CD4 + plus CD8 + cells) upon FasL stimulation was determined by their annexin V expression according to flow cytometry. Experiments were conducted independently three times. Representative histograms are shown.

Article Snippet: Naive CD4 + T cells (CD4 + CD62L + CD44 low ) were isolated from KO or WT mouse Spleen with EasySep mouse naive CD4 + T-cell isolation kits (19765, Stem Cell Technology).

Techniques: Staining, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Armc5 deletion causes developmental defects and compromises T-cell immune responses

doi: 10.1038/ncomms13834

Figure Lengend Snippet: ( a ) Proliferation of WT and KO naive spleen CD4 cells under Th1 and Th17 conditions was assessed based on CFSE content according to flow cytometry. Experiments were conducted three times, and representative histograms are shown. Grey peaks represent the CFSE content of CD4 cells at day 0. ( b ) These cells' differentiation into Th1 and Th17 cells was also determined by flow cytometry according to intracellular IFN-γ and IL-17 positivity (gated on total CD4 + ). Representative dot plots are shown in the left panel. Means±s.e.m. of data from three experiments are presented as bar graphs in the right panel. Mouse numbers ( n ) per group are indicated. P values are reported in the bar graphs (two-tailed Student's t -test). ( c , d ) T-bet and RORγt expression in CD4 cells cultured under Th1 and Th17 conditions or in IFNγ + or IL-17 + cells was determined by flow cytometry. Experiments were conducted three times. Representative histograms are shown. ( e ) Th1 and Th17 differentiation of naive spleen CD4 cells (CD45.2 single-positive) derived from WT and KO donors in chimeric mice was analysed by flow cytometry based on their intracellular IFN-γ and IL-17 expression. Representative dot plots are shown in the left panel. Means±s.e.m. of data from three experiments are presented as bar graphs in the right panel. Mouse numbers ( n ) per group are indicated. p values are reported in the bar graphs (two-tailed Student's t -test).

Article Snippet: Naive CD4 + T cells (CD4 + CD62L + CD44 low ) were isolated from KO or WT mouse Spleen with EasySep mouse naive CD4 + T-cell isolation kits (19765, Stem Cell Technology).

Techniques: Flow Cytometry, Two Tailed Test, Expressing, Cell Culture, Derivative Assay

Journal: Nature Communications

Article Title: Armc5 deletion causes developmental defects and compromises T-cell immune responses

doi: 10.1038/ncomms13834

Figure Lengend Snippet: ( a ) Means±s.e.m. of EAE clinical scores of KO and WT mice. * P <0.05 (two-tailed Student's t -test). ( b ) EAE incidence in KO and WT mice. * P <0.05 (chi-square test). ( c ) Means±s.e.m. of body weight of KO and WT mice during EAE induction. Body weight of mice on day 10 post-immunization was considered as 100%. * P <0.05 (two-tailed Student's t -test). ( d ) Means±s.e.m. of cellularity in draining LN and of cells infiltrating the CNS of mice 14 days after MOG immunization. Mouse numbers ( n ) and P values (paired two-tailed Student's t -test) are indicated. ( e , f ) Cytokine-producing cells among CD4 cells from draining LN ( e ) and CNS ( f ) on days 13–18 after MOG immunization. Left panels: representative dot plots; right panel: bar graphs (means±s.e.m.) summarizing all the results, with mouse numbers and P values (two-tailed Student's t -test) indicated. ( g ) HE (left column) or Luxol Fast Blue (right column) staining of spinal cords 30 days after MOG immunization. Asterisks indicate cell infiltration. Arrows point to demyelination. ( h ) Means±s.e.m. of mononuclear cell infiltration scores, demyelination scores and total pathological scores, which is the sum of the first two scores. Mouse numbers ( n ) and P values (two-tailed Student's t -test) are indicated. ( i ) Treg cells in naive KO mice on day 17 during EAE induction. Left panel: representative dot plots; right panel: means±s.e.m. of data from three experiments. NS: not significant (two-tailed Student's t -test).

Article Snippet: Naive CD4 + T cells (CD4 + CD62L + CD44 low ) were isolated from KO or WT mouse Spleen with EasySep mouse naive CD4 + T-cell isolation kits (19765, Stem Cell Technology).

Techniques: Two Tailed Test, Staining

Journal: Nature Communications

Article Title: Armc5 deletion causes developmental defects and compromises T-cell immune responses

doi: 10.1038/ncomms13834

Figure Lengend Snippet: ( a ) Means±s.e.m. of EAE clinical scores of chimeric mice.* P <0.05 (two-tailed Student's t -test). ( b ) EAE incidence in chimeric mice. * P <0.05 (chi-square test). ( c ) Means±s.e.m. of body weight of chimeric mice with body weight on day 10 after MOG immunization considered as 100%. No significant difference is found (two-tailed Student's t -test). ( d ) Cytokine-producing donor-derived CD4 cells in the CNS of chimeric mice on day 14 after MOG immunization. Left panel: representative dot plots; right panel: summary (means±s.e.m.) of all the results, with mouse numbers ( n ) and P -values (paired two-tailed Student's t -test) indicated.

Article Snippet: Naive CD4 + T cells (CD4 + CD62L + CD44 low ) were isolated from KO or WT mouse Spleen with EasySep mouse naive CD4 + T-cell isolation kits (19765, Stem Cell Technology).

Techniques: Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: Armc5 deletion causes developmental defects and compromises T-cell immune responses

doi: 10.1038/ncomms13834

Figure Lengend Snippet: ( a ) Absolute number of virus-specific, cytokine-producing CD8 cells. ( b , c ) Percentages of virus-specific, cytokine-producing cells among CD8 cells ( b ) and CD4 cells ( c ) on day 8 post LCMV infection. Means±s.e.m. of data are shown. Mouse numbers ( n ) per group and P values (two-tailed Student's t -test) are indicated. ( d ) Means±s.e.m. of percentages of gp 33–41 -specific CD107a+GranB+ CD8 T cells on day 8 post LCMV infection. Mouse numbers ( n ) per group and P values (two-tailed Student's t -test) are indicated. ( e ) Means±s.e.m. of viral titres in the kidney, liver and spleen on day 8 post LCMV infection. Mouse numbers ( n ) per group and P values (two-tailed Student's t -test) are indicated.

Article Snippet: Naive CD4 + T cells (CD4 + CD62L + CD44 low ) were isolated from KO or WT mouse Spleen with EasySep mouse naive CD4 + T-cell isolation kits (19765, Stem Cell Technology).

Techniques: Infection, Two Tailed Test