easysep mouse cd4 positive selection kit ii  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A) CD4 T-cells isolated from C57BL/6 mice were stimulated using anti-CD3 and anti-CD28 antibodies. Immunoblot shows Nrf2 and Keap1 protein expression in the lysates of cells harvested after the indicated number of days. β-actin is shown as the loading control. Data is representative of two independent experiments. (B) Box plot comparing the mRNA levels of Keap1 from Keap1-KO vs WT CD4 T-cells 48 h post-activation. Data are obtained from RNAseq analysis (n=3). (C) Representative histogram overlay and summary graph show Nrf2 protein expression in CD4 T-cells isolated from WT and Keap1-KO mice on day 2 after injection with anti-CD3 antibody intraperitoneally (n=2). (D) Flow cytometric plot showing the FSC and SSC of CD4 T-cells activated in vitr o. Data is representative of three independent experiments. (E) Representative histogram overlay showing proliferation of WT, Keap1-KO, and Nrf2-KO CD4 T-cells stimulated in vitro for 72 hours. Data is representative of three different experiments. (F) A representative gating strategy of flow cytometric analysis of data shown in . (G) Volcano plot showing the significantly upregulated (red) and downregulated (blue) genes identified from RNA-seq analysis of Keap1-KO vs WT CD4 T-cells 48 h post-activation. (n=3). (H) Gene Ontology data showing the upregulated genes involved in the biological processes in Keap1-KO over WT 48 h post-activation. Data are obtained from RNA-seq analysis (n=3). (I) Box plot comparing the mRNA levels of the indicated genes between Keap1-KO vs WT CD4 T-cells 48 h post-activation. Data are obtained from RNAseq analysis. Data are shown as mean ± SEM from the indicated number of sets of mice. *p<0.05, **p<0.01, ****p<0.0001

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: Isolation, Western Blot, Expressing, Activation Assay, Injection, In Vitro, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A) An illustration depicting the in vitro activation assay of naïve CD4 + T-cells using anti-CD3 and anti-CD28 antibodies, resulting in T-cell expansion. (B) Naïve CD4 + T-cells isolated from Keap1-KO and wild-type (WT) littermate mice were labeled with Cell Trace dye and activated in vitro for 96 h. A representative histogram overlay shows the dye dilution, and the graph shows the summary of proliferation (n=4-5). (C) Representative histogram overlay comparing the cell size (FSC, forward scatter) of WT and Keap1-KO CD4 + T-cells 96h post-activation. (D) Schematic diagram of in vivo BrdU incorporation assay after injecting anti-CD3 antibodies into the indicated mice. (E) Summary graphs of percentages of BrdU + CD4 + T-cells within mesenteric lymph nodes of WT, Keap1-KO, and Nrf2-KO mice treated as in (D) (n=3-6). (F) Schematic diagram of in vivo BrdU incorporation assay in ova-TCR transgenic (OTII)-WT, OTII-Keap1-KO, and OTII-Nrf2-KO mice after antigen challenge with ovalbumin. (G) Percentages of splenic BrdU + CD4 + T-cells in indicated OTII mice treated as in (F) (n=4-6). (H) Total splenocytes from OTII-WT and OTII-Keap1-KO mice were cultured in vitro with ova peptide in the presence of BrdU. Histogram overlay and graph shows percentages of BrdU + CD4 + Vβ5.1 + T-cells (n=3-4). (I) Daily cell counts of in vitro activated WT, Keap1-KO, and Nrf2-KO CD4 + T-cells were measured (n=7-11). (J) Representative histogram overlay and summary data of Apotracker green staining in CD4 + T-cells 72h post-activation in vitro (n=3 or 5). All data are shown as mean ± SEM from the indicated number of sets of mice. *p<0.05, **p<0.01, ****p<0.0001. K-KO: Keap1-KO, N-KO: Nrf2-KO

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: In Vitro, Activation Assay, Isolation, Labeling, In Vivo, BrdU Incorporation Assay, Transgenic Assay, Cell Culture, Staining

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A, B) The expression of (A) CD44 (n=8) and (B) CD25 (n=5) was measured in naïve CD4 + T-cells from WT, Keap1-KO, and Nrf2-KO mice activated in vitro as in . (C, D) OTII-WT, OTII-Keap1-KO, and OTII-Nrf2-KO mice were challenged with ovalbumin in vivo . The expression of (C) CD44, and (D) CD25 in CD4 + Vβ5.1 + (OTII) cells was measured (n=4-5). (E-G) The expression of Nur77 (E) after 4h, and phosphorylated Zap-70 (F) and ERK (G) after 24h was compared between WT, Keap1-KO, and Nrf2-KO CD4 + T-cells activated in vitro (n=3-6). (H) Heatmap of RNAseq analysis comparing the gene expression levels in Keap1-KO vs WT naïve CD4 + T-cells 24h post-activation (n=3). (I) Relative gene expression levels of IL-2 and IL-2ra in WT and Keap1-KO CD4 + T-cells 24h post-activation measured by qRT-PCR. Data are representative of three independent experiments. (J) Secreted IL-2 protein levels from the culture supernatants of WT, Keap1-KO, and Nrf2-KO CD4 + T-cells 48h post-activation were measured by ELISA (n=3). (K, L) Phosphorylation levels of STAT5 (K) and S6 proteins in WT, Keap1-KO, and Nrf2-KO CD4 + T-cells 24h post-activation are shown (n=5-7). Histogram overlays show representative data and graphs show mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: Expressing, In Vitro, In Vivo, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A) Volcano plot showing the significantly upregulated (red) and downregulated (blue) genes in Keap1-KO vs WT CD4 T-cells 24h post-activation identified from RNA-seq analysis. (n=3). (B) Gene Ontology enrichment analysis of molecular functions for upregulated genes between Keap1KO vs WT CD4 T-cells 48h post activation. Data are obtained from RNA-seq analysis (n=3). (C) Representative histogram overlay and summary graph show the levels of phosphorylated NF-kB in WT, Keap1-KO, and Nrf2-KO CD4 T-cells activated in vitro for 24 h (n=4) Data are shown as mean ± SEM from the indicated number of sets of mice.

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: Activation Assay, RNA Sequencing Assay, In Vitro

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A) Representative histogram overlay of the proliferation of WT, Keap1-KO, and Double-KO mice. Data is representative of three different experiments. (B) Intracellular levels of pyruvate were compared between WT, Keap1-KO, and Double-KO mice CD4 T-cells activated in vitro for 48 h (n=3). (C) Glucose uptake of WT, Keap1-KO, and Double-KO mice CD4 T-cells was measured 48h after activation by incubating with 2-NBDG for 30 mins followed by flow cytometry. Representative histogram overlay and summary graph are shown (n=3). (D) Box plots from RNAseq analysis comparing the mRNA levels of the indicated genes between Keap1-KO vs WT CD4 T-cells 48 h post-activation. Data are shown as mean ± SEM from the indicated number of sets of mice.

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: In Vitro, Activation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A) Levels of intracellular and extracellular lactate from WT, Keap1-KO, and Double-KO CD4 + T-cells were measured 48h post-activation in vitro (n=3). (B-E) Naïve CD4 + T-cells from WT, Keap1-KO, and Double-KO mice were activated in vitro for 48h and subjected to a glycolytic stress test. The representative graphs show ECAR over time (B), glycolysis (C), glycolytic capacity (D), and glycolytic ATP (E) (n=3). (F-H) A glycolytic stress test was performed using OTII-WT and OTII-Keap1-KO CD4 + T-cells isolated from mice 72h after the ova challenge. ECAR over time (F), glycolysis (G), and glycolytic capacity (H) are shown. Data is representative of two independent experiments. (I) Representative histogram overlay and summary of the proliferation of WT and Keap1-KO CD4 + T-cells activated in vitro in media with 11mM glucose ( upper panel ) or without glucose ( lower panel) (n=4). (J) The ratio of indicated metabolites in Keap1-KO over WT CD4 + T-cells activated in vitro for 72 h followed by CE-MS analysis (n=2). Data are shown as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. K-KO: Keap1-KO

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: Activation Assay, In Vitro, Isolation

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A-C) The Levels of glutamine (A), glutamate (B), and a-ketoglutarate (C) from WT, Keap1-KO, and Double-KO naïve CD4 + T-cells were measured 48h after in vitro stimulation (n=5-6) (D) The percentage contributions of 13 C-labeled glutamine (M1+) and 12 C Glucose (M0) to the total metabolites isolated from activated WT and Keap1-KO CD4 + T-cells are shown. (n=2). (E) Graph shows the % pool of Citrate from 13 C-labeled glutamine in WT, Keap1-KO CD4 + T-cells 72 h post-activation (n=2). (F-G) Representative plots show the proliferation (A) and the cell size (G) of WT and Keap1-KO CD4 + T-cells activated in vitro in media containing limiting glutamine (0.2mM or 0.02mM) and glucose (n=4). (H) WT and Keap1-KO CD4 + T-cells were treated with Vehicle or 250nM CB-839 for xx h during in vitro activation. Summary graphs compare the cell counts (n=4). (I) Intracellular ATP levels were compared between WT and Keap1-KO CD4 + T-cells activated in media with glucose and 2mM or 0.02mM glutamine (n=3). Data are shown as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. K-KO: Keap1-KO, Gln: glutamine, Glc: glucose

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: In Vitro, Labeling, Isolation, Activation Assay

Journal: bioRxiv

Article Title: Nrf2 regulates the activation-driven expansion of CD4 + T-cells by differentially modulating glucose and glutamine metabolism

doi: 10.1101/2024.04.18.590146

Figure Lengend Snippet: (A) Microscopic Images demonstrate the differences in cell morphology between WT and Keap1-KO CD4 T-cells activated in vitro for 96h in the presence of vehicle or CB-839. Data are representative of three independent experiments. (B) Representative histogram overlays showing the expression of CD25, CD44, and CD69 in WT and Keap1-KO CD4 T-cells activated in vitro in media with limiting glutamine (Gln). Data is representative of three independent experiments. (C) Summary graph showing cell proliferation of WT and Keap1-KO CD4 T-cells treated with Vehicle or CB-839 (n=3). Data are shown as mean ± SEM from the indicated number of sets of mice.

Article Snippet: The total CD4 T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (STEMCELL Technologies).

Techniques: In Vitro, Expressing

Journal: Nature immunology

Article Title: TCF-1 promotes chromatin interactions across topologically associating domains in T cell progenitors

doi: 10.1038/s41590-022-01232-z

Figure Lengend Snippet: REAGENT or RESOURCE

Article Snippet: EasySep TM Mouse CD4 Positive Selection Kit II , Stemcell , Cat# 18952.

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Magnetic Beads, Purification, Saline, Electron Microscopy, Library Amplification, Library Quantification, Selection, Plasmid Preparation, HiChIP, Software