Cd4 t cells

Manufactured by STEMCELL

Sourced in Canada

CD4 T cells are a type of lymphocyte that express the CD4 protein on their cell surface. They play a crucial role in the adaptive immune response by helping to coordinate and regulate various immune functions.

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Lab products found in correlation

Lsrfortessa x 20 instrument, by BD (2 mentions) Zombie aqua fixable viability kit, by BioLegend (2 mentions) Violet proliferation dye, by BD (1 mentions) Cd3 apc cy7, by BioLegend (1 mentions) Cd19 pe, by BioLegend (1 mentions) B cell enrichment kit, by STEMCELL (1 mentions) Cd4 pe cy7 sk3, by BioLegend (1 mentions) Ifn γ, by Santa Cruz Biotechnology (1 mentions) Il 17, by Santa Cruz Biotechnology (1 mentions) Facsaria 2, by BD (1 mentions) Anti cd3, by BioLegend (1 mentions) Cytofix fixation buffer, by BD (1 mentions) Perm buffer 3, by BD (1 mentions)

Close spelling variants of this product

Human CD4+ T cells EasySep CD4+ T cells Enrichment Kit RosetteSep Human CD4+ T Cells Enrichment Cocktail RCD4 (CD3+/CD4+/CD69-/CD25-/HLA-DR-) T cells CD4+ T cells isolation kit EasySep Human CD4+ T cells Enrichment Kit

8 protocols using cd4 t cells

Assessing CD4+ T Cell Proliferation

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Prior to stimulation for B10 cells, B cells were negatively isolated from a starting population of 10⁷ PBMCs, using the B cell enrichment kit (Stemcell). Isolated B cells were plated in 96-well flat bottom plates in RPMI + 10% FBS. Cells were stimulated with CpG and rCD40L for 48 h at 37°C in 5% CO₂ incubator. At 43 h, cells were restimulated with PMA and ionomycin. In parallel with the end of the 48-h stimulation, PBMCs from the same patients were enriched for CD4 T cells (Stemcell) and stained with the Violet Proliferation dye (1 µM, BD Bioscience) in PBS and incubated in a 37°C water bath for 15 min. After 48 h, the isolated CD4 cells were combined with B cells at 1:1 and 1:2 ratio of T:B cells along with a CD4 T cell only condition and stimulated with αCD3/αCD28 for 5 days. For experiments using the transwell plates, stimulated B cells were plated in top chamber of 24-well transwell plates while CD4 T cells and αCD3/αCD28 were plated in bottom chamber.
To visualize proliferation of the CD4 T cells, cells were stained with Zombie Violet, CD14 Brilliant Violet 510 (M5E2, Biolegend), CXCR5 AlexaFluor 647 (RF8B2, BDB), CD8 AlexaFluor 700 (SK1, Biolegend), CD3 APC-Cy7 (SK7, Biolegend), CD19 PE (HIB19, Biolegend), and CD4 PE-Cy7 (SK3, Biolegend) conjugate for 25 min at 4°C. Following cell surface staining, cells were fixed with 1% PFA and acquired on a LSRII flow cytometer (BDB).

Yi J.S., Russo M.A., Massey J.M., Juel V., Hobson-Webb L.D., Gable K., Raja S.M., Balderson K., Weinhold K.J, & Guptill J.T. (2017). B10 Cell Frequencies and Suppressive Capacity in Myasthenia Gravis Are Associated with Disease Severity. Frontiers in Neurology, 8, 34.

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Isolation and Stimulation of Primary Mouse T Cells

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Primary mouse CD4⁺ T cells (Cat#19852, STEMCELL Technologies, Vancouver, Canada) from dLNs during EAE or CD4⁺ T (Cat#19852, STEMCELL Technologies, Vancouver, Canada), CD8⁺ T (Cat#19853, STEMCELL Technologies, Vancouver, Canada), and CD11b⁺ myeloid (Cat#18970, STEMCELL Technologies, Vancouver, Canada) cells from spleen and peripheral lymph nodes at steady state were purified following manufacturer’s instructions, after mashing through a 40-μm nylon filter (Cat#22363547, Thermo Fisher Scientific, PA, USA). Isolated CD4⁺ T cells were stimulated at 37 °C and 5% CO₂ for 30 min in 10% RPMI containing PMA (20 ng/ml) and Ionomycin (1 µg/ml) for Malt1 and Stat3 ubiquitination analysis.

Cho J.J., Xu Z., Parthasarathy U., Drashansky T.T., Helm E.Y., Zuniga A.N., Lorentsen K.J., Mansouri S., Cho J.Y., Edelmann M.J., Duong D.M., Gehring T., Seeholzer T., Krappmann D., Uddin M.N., Califano D., Wang R.L., Jin L., Li H., Lv D., Zhou D., Zhou L, & Avram D. (2019). Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation. Nature Communications, 10, 701.

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Modulation of CD4+ T cell responses by H. pylori and IL-23

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In a 5-day incubation, purified human peripheral or mouse spleen CD4⁺ T cells (StemCell Technologies) were cocultured (2×10⁵ cells/well) with WT H. pylori or ΔcagA stimulated-DCs from autologous blood; or WT H. pylori or ΔcagA stimulated-bone marrow–derived dendritic cells (BMDCs) from WT or IL-23 KO mice at 2:1 ratio. Alternatively, CD4⁺ T cells were cocultured with autologous ΔcagA-stimulated DCs at 2:1 ratio supplemented with IL-23 (10 ng/mL) or media alone, or with autologous WT H. pylori-stimulated DCs at 2:1 ratio supplemented with IL-23 Ab (10 μg/mL) or control IgG (10 μg/mL). CD4⁺ T cells were also first labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cocultured (1×10⁵ cells/well) with MDSCs at different ratios. After such 5-d incubation, cells were collected and analysed by intracellular cytokine staining.

Zhuang Y., Cheng P., Liu X.F., Peng L.S., Li B.S., Wang T.T., Chen N., Li W.H., Shi Y., Chen W., Pang K.C., Zeng M., Mao X.H., Yang S.M., Guo H., Guo G., Liu T., Zuo Q.F., Yang H.J., Yang L.Y., Mao F.Y., Lv Y.P, & Zou Q.M. (2014). A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis. Gut, 64(9), 1368-1378.

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Phosphorylation Analysis of Immune Cells

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Phosphorylation status of p65 and c-JUN were analyzed by flow cytometry in isolated CD4 T cells (StemCell Technologies) as previously described (33 (link)). Briefly, cells were stimulated for 15 minutes at 37°C in the absence or presence of 2.5µM ALN, 2.5µg/mL PAM, 1µg/mL ZOL or 50nM PMA as a positive control. Cells were washed and stained with a viability dye (Zombie Aqua Fixable viability kit, BioLegend) and fixed with 100µl of pre-warmed Cytofix Fixation Buffer (BD Biosciences) for 10 minutes at 37°C. Cells were permeabilized while vortexing with 100 µL of prechilled Perm Buffer III (BD Biosciences) and incubated for 30 minutes on ice. After incubation, cells were washed and stained with anti-NFkB p65 (pS529, clone K10-895.12.50, BD Biosciences) or Phospho-c-Jun (Ser73, clone D47G9, Cell Signaling Technology) for 16 hours at 4°C. After washing, samples were acquired on a BD LSR Fortessa TM X-20 instrument (BD Biosciences) and analyzed using Flowjo (FlowJo v.10.8.1).

Sanz M., Weideman A.M., Ward A.R., Clohosey M.L., Garcia-Recio S., Selitsky S.R., Mann B.T., Iannone M.A., Whitworth C.P., Chitrakar A., Garrido C., Kirchherr J., Coffey A.R., Tsai Y.H., Samir S., Xu Y., Copertino D., Bosque A., Jones B.R., Parker J.S., Hudgens M.G., Goonetilleke N, & Soriano-Sarabia N. (2023). Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1. Frontiers in Immunology, 14, 1219250.

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Isolation and Analysis of CD4+ T Cells

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Peripheral blood was collected into heparinized tubes, diluted to 1.5 ml final volume with PBS, and stored at 4°C for <4 h. Samples were layered onto Ficoll Paque Plus, and peripheral blood mononuclear cells were isolated by using a density gradient separation technique. An automated magnetic bead–based positive selection protocol was used to isolate CD4⁺ T cells (Stemcell Technologies, Vancouver, BC, Canada). All Western blot analyses were then performed according to standardized protocols above described. Here, primary Abs used were as follows: IL-17 (1:250; Santa Cruz Biotechnology) and IFN-γ (1:250; Santa Cruz Biotechnology). Representative blot images from 3 separate analyses are given.

Giacoppo S., Thangavelu S.R., Diomede F., Bramanti P., Conti P., Trubiani O, & Mazzon E. (2017). Anti-inflammatory effects of hypoxia-preconditioned human periodontal ligament cell secretome in an experimental model of multiple sclerosis: a key role of IL-37. The FASEB Journal, 31(12), 5592-5608.

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Adoptive Transfer of exTreg Attenuates Atherosclerosis

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Cells from lymph nodes and spleens from pooled p6- or MOG-immunized lineage tracker mice were extracted and enriched for CD4 T cells (StemCell). exTreg were sorted by FACS ARIA II (BD Biosciences) gating on lymphocyte morphology, single cells and live cells (DAPI⁻)CD4⁺TCRb⁺GFP⁻RFP⁺. Anti-mouse CD4 antibody (clone GK1.5, Biolegend), and anti-mouse TCRβ antibody (clone H57-597, Biolegend) were used. 2.10⁵ exTreg were injected retro-orbitally in 100uL of PBS 1X in 8-10 weeks old 6Gy irradiated female Apoe^−/− mice that had been on WD (WD; 42% kcal from fat, 0.2% cholesterol) for 3 weeks and antibiotics 3 days before and for 14 days. The other mice were injected with 100uL of PBS as negative control. Recipient mice continued on WD for 5 more weeks. At the end of the experiment, blood and aortas were harvested to determine engraftment, plasma IFNγ (Invitrogen ELISA) and atherosclerotic lesions (Sudan-IV), respectively.

Saigusa R., Roy P., Freuchet A., Gulati R., Ghosheh Y., Suthahar S.S., Durant C.P., Hanna D.B., Kiosses W.B., Orecchioni M., Wen L., Wu R., Kuniholm M.H., Landay A.L., Anastos K., Tien P.C., Gange S.J., Kassaye S., Vallejo J., Hedrick C.C., Kwok W.W., Sette A., Hodis H.N., Kaplan R.C, & Ley K. (2022). Single cell transcriptomics and TCR reconstruction reveal CD4 T cell response to MHC-II-restricted APOB epitope in human cardiovascular disease. Nature cardiovascular research, 1(5), 462-475.

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Induction of IL-10-Producing Tr1 Cells

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Peripheral Blood Mononuclear Cel (PBMCl) were isolated from blood obtained after informed consent of patients in the relapsing-remitting stage (RRMS, see patient description in Table 1, ethical approval SR258) or from age and sex-matched healthy donors (ethical approval AMREC 115-HV-013), and samples were obtained after informed consent according to the Declaration of Helsinki. Purified CD4+ T cells (StemCell, Grenoble, France) (Supplementary Figure S1) were activated for four days with pre-coated anti-CD3 (OKT3, 5µg/ml)/anti-CD46 (MC120.6, 10 µg/ml) or anti-CD3/anti-CD28 (CD28.2, Biolegend, 5 µg/ml), and 10 U/ml of rhIL-2 (Cambridge Bioscience, Cambridge, UK) (13 (link)). At the beginning of the culture, cells were treated with 10^-7M 1,25(OH)2D3 (Sigma, Gillingham, UK), a dose previously reported to promote IL-10-producing Tr1 cells (6 (link), 32 (link), 33 (link)) or the vehicle control, ethanol.

Killick J., Hay J., Morandi E., Vermeren S., Kari S., Angles T., Williams A., Damoiseaux J, & Astier A.L. (2020). Vitamin D/CD46 Crosstalk in Human T Cells in Multiple Sclerosis. Frontiers in Immunology, 11, 598727.

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Phosphorylation Status of p65 and c-JUN in Activated CD4 T Cells

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Phosphorylation status of p65 and c-JUN were analyzed by flow cytometry in isolated CD4 T cells (StemCell Technologies) as previously described (44 (link)). Briefly, cells were stimulated for 15 minutes at 37°C in the absence or presence of 2.5μM ALN, 2.5μg/mL PAM, 1μg/mL ZOL or 50nM PMA as a positive control. Cells were washed and stained with a viability dye (Zombie Aqua Fixable viability kit, BioLegend) and fixed with 100μl of pre-warmed Cytofix Fixation Buffer (BD Biosciences) for 10 minutes at 37°C. Cells were permeabilized while vortexing with 100 μL of prechilled Perm Buffer III (BD Biosciences) and incubated for 30 minutes on ice. After incubation, cells were washed and stained with anti-NFkB p65 (pS529, clone K10–895.12.50, BD Biosciences) or Phospho-c-Jun (Ser73, clone D47G9, Cell Signaling Technology) for 16 hours at 4°C. After washing, samples were acquired on a BD LSR Fortessa TM X-20 instrument (BD Biosciences) and analyzed using Flowjo (FlowJo v.10.8.1).

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