cd4 t cell isolation kit  (Miltenyi Biotec)

Journal: Science Advances

Article Title: Pyruvate kinase muscle 2 (PKM2) promotes CD4 T cell survival by regulating pyruvate oxidation during homeostasis and expansion

doi: 10.1126/sciadv.aec5092

Figure Lengend Snippet: CD4 T cells purified from a pool of spleen and lymph node cells were expanded with anti-CD3/CD28 in the presence of TEPP-46 or DMSO control. After 96 hours, cells were analyzed by flow cytometry to determine; ( A ) number of live (Sytox − ) cells; ( B ) cell proliferation determined by CellTrace Violet (CTV) dilution; ( C ) effects of IL-7 and IL-2 on cell numbers; ( D ) the level of DCFDA staining; ( E ) effects of NAC on cell number; ( F ) effects of atmospheric (17% O 2 ) or 3% O 2 on cell numbers. All data in this figure represents three or more independent experiments. Graphs represent means ± SEM, statistical significance was measured by one-way ANOVA test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. n.s., not significant.

Article Snippet: CD4 T cells were purified from pooled spleen and lymph node cell suspensions using CD4 microbeads (positive selection), CD4 T cell isolation kit (negative selection), or naïve CD4 T cell isolation kit all purchased from Miltenyi Biotec and used according to the manufacturer’s protocol.

Techniques: Purification, Control, Flow Cytometry, Staining

Journal: Science Advances

Article Title: Pyruvate kinase muscle 2 (PKM2) promotes CD4 T cell survival by regulating pyruvate oxidation during homeostasis and expansion

doi: 10.1126/sciadv.aec5092

Figure Lengend Snippet: ( A ) Thymus serial histological sections from PKM2 fl/fl (control) and PKM2 fl/fl CD4 cre (PKM2 ΔTcell ) mice stained for PKM1, PKM2, and GLUT1. ( B ) PKM1 and PKM2 protein expression of flow sorted thymic SP4 and SP8 cells. ( C to G ) Flow cytometry analysis was performed on isolated control and CD4 T cells ΔPKM2 expanded for 96 hours with CD3/CD28 to determine; the number of live (Sytox − ) (C) and dead (Sytox + ) (D) cells; cell proliferation using CTV dilution (E); DCFDA staining intensity (F); MitoSOX red staining intensity at indicated times (G). ( H ) Isolated control and CD4 T cells ΔPKM2 were activated with CD3/CD28 and after 24 hours MitoTEMPO was added at indicated concentration, and the percent of live cells were determined by flow cytometry at 72 hours. ( I and J ) Isolated control and PKM2 deficient CD4 T cells were activated with CD3/CD28 under 1 (I) or 3% (J) oxygen for 96 hours, and the number of live and dead cells was determined by flow cytometry. (A) Representative of three individual mice. (B) Representative from two independent experiments. [(C) to (I)] Representative of three or more independent experiments. Graphs represent mean SEM; statistical significance was measured by two-way ANOVA multiple-comparisons test for (C), (D), and (F) to (I). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. n.s., not significant.

Article Snippet: CD4 T cells were purified from pooled spleen and lymph node cell suspensions using CD4 microbeads (positive selection), CD4 T cell isolation kit (negative selection), or naïve CD4 T cell isolation kit all purchased from Miltenyi Biotec and used according to the manufacturer’s protocol.

Techniques: Control, Staining, Expressing, Flow Cytometry, Isolation, Concentration Assay

Journal: Science Advances

Article Title: Pyruvate kinase muscle 2 (PKM2) promotes CD4 T cell survival by regulating pyruvate oxidation during homeostasis and expansion

doi: 10.1126/sciadv.aec5092

Figure Lengend Snippet: ( A and B ) CD4 + T cells were isolated from control and PKM2 ΔTcell mice and then expanded with anti-CD3/CD28 for 96 hours. Glycolytic activity was measured as extracellular acidification rate (ECAR) following sequential injections of rotenone, antimycin, and 2-deoxyglucose (2-DG) (A). Mitochondrial respiration (OXPHOS) was assessed by OCR after sequential injections of oligomycin, BAM15, rotenone, and antimycin (B). Data in (A) and (B) are representative of at least three independent experiments. ( C to E ) CD4 + T cells from control and PKM2 ΔTcell mice were expanded for 72 hours with anti-CD3/CD28. Heatmap showing normalized expression levels of targeted glycolytic and TCA metabolites (C). Schematic illustrating [U- 13 C] glucose–labeling patterns after metabolism through glycolysis and the TCA cycle (open circles, 12 C; blue-filled circles, 13 C) (D). Following 72 hours of expansion, cells were pulsed for 4 hours with [U- 13 C] glucose, and isotopolog tracing was performed to quantify carbon flow into glycolytic and TCA intermediates (E). For (C) and (E), samples were obtained from three individual mice per genotype. Data are presented as means ± SEM. Statistical significance was determined using the Mann-Whitney U test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: CD4 T cells were purified from pooled spleen and lymph node cell suspensions using CD4 microbeads (positive selection), CD4 T cell isolation kit (negative selection), or naïve CD4 T cell isolation kit all purchased from Miltenyi Biotec and used according to the manufacturer’s protocol.

Techniques: Isolation, Control, Activity Assay, Expressing, Labeling, MANN-WHITNEY

Journal: Science Advances

Article Title: Pyruvate kinase muscle 2 (PKM2) promotes CD4 T cell survival by regulating pyruvate oxidation during homeostasis and expansion

doi: 10.1126/sciadv.aec5092

Figure Lengend Snippet: ( A ) Pyruvate kinase activity of lysates from CD4 + T cells isolated from control or PKM2 ΔTcell mice. OD 570 , optical density at 570 nm. ( B ) Flow cytometric analysis of spleens from aging control and PKM2 ΔTcell mice ( n ≥ 5 per age group), showing CD4:CD8 T cell ratio (left), CD4 + T cell frequency (middle), and absolute CD4 + T cell numbers (right). ( C and D ) Flow cytometry analysis on thymus, spleen, and lymph nodes of 15-week-old control and PKM2 ΔTcell mice ( n = 5 per group) for the frequency of CD8 + T cells (C) and CD4:CD8 T cell ratio and CD4 + T cell frequency (D). ( E ) Flow cytometry gating strategy for CD4 + T cell subsets: naïve (CD44 low CD62L high ), central memory (CM; CD44 high CD62L high ), effector memory (EM; CD44 high CD62L low ), and effector (eff; CD44 low CD62L low ). ( F ) Frequencies of CD4 + T cell memory subsets in spleens from control and PKM2 ΔTcell mice at indicated ages ( n ≥ 5 per group). ( G ) Schematic of mixed bone marrow (BM) chimera experiment: lethally irradiated RAG (−/−) mice reconstituted with a 1:1 mixture of BM cells from CD45.1 + wild-type donors ( n = 6) and PKM2 ΔTcell donors ( n = 7). D0, day 0; 10W, 10 weeks. ( H ) Flow cytometric analysis of chimeras at 10 weeks postreconstitution. Data from spleens and lymph nodes are shown as CD4:CD8 T cell ratio (top row), percentage of CD4 + T cells (middle row), and percentage of CD8 + T cells (bottom row). Spleen data combine three independent experiments; lymph node data: CD45.1 ( n = 6) and PKM2 ΔTcell ( n = 7). (A) Means ± SEM; significance by simple linear regression. [(B) to (D), (F), and (H)] Means ± SEM; significance by Mann-Whitney U test. * P < 0.05; ** P < 0.01;*** P < 0.001; **** P < 0.0001. n.s., not significant.

Article Snippet: CD4 T cells were purified from pooled spleen and lymph node cell suspensions using CD4 microbeads (positive selection), CD4 T cell isolation kit (negative selection), or naïve CD4 T cell isolation kit all purchased from Miltenyi Biotec and used according to the manufacturer’s protocol.

Techniques: Activity Assay, Isolation, Control, Flow Cytometry, Irradiation, MANN-WHITNEY

Journal: Science Advances

Article Title: Pyruvate kinase muscle 2 (PKM2) promotes CD4 T cell survival by regulating pyruvate oxidation during homeostasis and expansion

doi: 10.1126/sciadv.aec5092

Figure Lengend Snippet: ( A and B ) Thymectomy (T) ( n = 7) or sham surgery (S) ( n = 3) was performed on control and PKM2 ΔTcell mice. After 10 weeks, flow cytometric analysis to determine the mean fold change (MFC) in CD4:CD8 T cell ratio (left column), number of CD4 T cells (middle column), and frequency of CD4 T cells (right column) from cell suspensions taken from spleen (A) and lymph nodes (B) of control and PKM2 ΔTcell mice. ( C ) Naïve CD4 T cells isolated from control (CD45.1) and PKM2 ΔTcell (CD45.2) mice were labeled with CTV, and a known ratio of cells was intravenously injected into irradiated B6 mice. After 7 days, spleen and lymph node CD4 T cell proliferation was determined by CTV dilution using flow cytometry. ( D ) Naïve CD4 T cells isolated from control (CD45.1) and PKM2 ΔTcell (CD45.2) mice were injected in equal ratio (donor cells) into RAG (−/−) mice (day 0). After 21 days, the frequency of control and CD4 T cells ΔPKM2 was examined by flow cytometry in spleen and lymph nodes. Graphs represent ±SEM; statistical significance was measured by two-way ANOVA multiple-comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. n.s., not significant.

Article Snippet: CD4 T cells were purified from pooled spleen and lymph node cell suspensions using CD4 microbeads (positive selection), CD4 T cell isolation kit (negative selection), or naïve CD4 T cell isolation kit all purchased from Miltenyi Biotec and used according to the manufacturer’s protocol.

Techniques: Control, Isolation, Labeling, Injection, Irradiation, Flow Cytometry

Journal: Science Advances

Article Title: Pyruvate kinase muscle 2 (PKM2) promotes CD4 T cell survival by regulating pyruvate oxidation during homeostasis and expansion

doi: 10.1126/sciadv.aec5092

Figure Lengend Snippet: ( A ) Schematic of the experimental colitis model: CD4 + CD45RB hi T cells were purified from either control or PKM2 ΔTcell mice and intraperitoneally transferred into RAG (− / −) recipients. ( B ) Body weight changes over the course of the experiment. ( C ) Analysis of mesenteric lymph node (MLN) cells on day 51 for frequency of Foxp3 + regulatory T cells within CD4 + CD3 + T cells. ( D to G ) MLN cells stimulated for 5 hours with PMA and ionomycin in the presence of brefeldin A and monensin to detect the percentage of T H 17 cells (IL-17 + ) (D), pathogenic T H 17 cells (IL-17 + IFN-γ + ) (E), T H 1 cells (IFN-γ + ) (F), and T H 2 cells (IL-4 + ) (G). ( H to J ) Analysis of MLN T cell populations at day 51 to determine total MLN cell numbers (H), proportion of CD4 + T cells (I), and absolute number of CD4 + T cells (J). ( K ) Representative immunohistochemical staining for CD4 in colon sections. ( L ) Quantification of CD4 signal intensity. ( M ) Area of colon tissue positive for CD4 staining. ( N ) Representative hematoxylin and eosin (H&E)–stained colon sections. ( O ) Histopathological disease severity scores, based on structural analysis (methods detailed in Materials and Methods). Statistics: (B) nonlinear Padé approximation curve fit with 95% confidence intervals (dotted lines). [(C) to (J)] Unpaired t test. Results are presented as means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. n.s., not significant.

Article Snippet: CD4 T cells were purified from pooled spleen and lymph node cell suspensions using CD4 microbeads (positive selection), CD4 T cell isolation kit (negative selection), or naïve CD4 T cell isolation kit all purchased from Miltenyi Biotec and used according to the manufacturer’s protocol.

Techniques: Purification, Control, Immunohistochemical staining, Staining