stem cell easysep mouse t cell isolation kit  (STEMCELL Technologies Inc)

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: CD8 + T cells were isolated from the spleen of naive or infected WT mice, with EasySep mouse CD8 + T-cell isolation kits (19853, 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: CD8 + T cells were isolated from the spleen of naive or infected WT mice, with EasySep mouse CD8 + T-cell isolation kits (19853, 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 ) Spleen CD8 cell numbers in KO mice on day 8 after LCMV infection as determined by flow cytometry. Mice number ( n ), means±s.e.m. and P values (two-tailed Student's t -test) are indicated. ( b ) Virus-specific spleen CD8 cells in KO mice on day 8 post-LCMV infection according to flow cytometry. Representative dot plots are shown. ( c ) Means±s.e.m. of percentages of gp33–41, np396–405 and gp276–286 tetramer-positive cells among spleen CD8 cells from all the results are presented. Numbers ( n ) of mice per group and P values (two-tailed Student's t -test) are indicated. ( d ) Means±s.e.m. of absolute numbers of gp33–41, np396–405 and gp276–286 tetramer-positive CD8 cells in the KO and WT mouse spleens on day 8 post infection. Numbers ( n ) of mice per group and P values (two-tailed Student's t -test) are indicated. ( e , f ) Memory and effector CD8 cell maturation in LCMV-infected WT and KO mice on day 8 post LCMV infection. KLRG1loCD127hi cells are considered as memory precursor effector cells (MPEC), and KLRG1hiCD127lo cells as short-lived effector cells (SLEC). Means±s.e.m. are presented. Numbers ( n ) of mice per group and P values (two-tailed Student's t -test) are indicated ( e ). Representative dot plots are shown ( f ). ( g ) On day 8 post infection, total gp33–41 np396–405 and gp276–286 tetramer-positive CD8 cells in KO and WT mouse spleen were assessed for activation markers. Means±s.e.m. are presented. Numbers ( n ) of mice per group and P values (two-tailed Student's t -test) are indicated.

Article Snippet: CD8 + T cells were isolated from the spleen of naive or infected WT mice, with EasySep mouse CD8 + T-cell isolation kits (19853, Stem Cell Technology).

Techniques: Infection, Flow Cytometry, Two Tailed Test, Activation 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: CD8 + T cells were isolated from the spleen of naive or infected WT mice, with EasySep mouse CD8 + T-cell isolation kits (19853, Stem Cell Technology).

Techniques: Infection, Two Tailed Test

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