Human cd4 t cell isolation kit

Manufactured by STEMCELL

Sourced in United States, Canada

The Human CD4+ T cell isolation kit is a product designed to isolate CD4+ T cells from human samples. It utilizes a fast and efficient process to enrich for CD4+ T cells, allowing for their subsequent analysis or experimentation.

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Lab products found in correlation

Texmacs gmp medium, by Miltenyi Biotec (2 mentions) Clinimacs plus system, by Miltenyi Biotec (2 mentions) Interleukin 2 (il 2), by Merck Group (1 mentions) Anti cd3, by BD (1 mentions) Anti cd28, by BD (1 mentions) Rhgal 3, by R&D Systems (1 mentions) Rhgal 9, by R&D Systems (1 mentions) Facsaria 2, by BD (1 mentions) Mouse cd4 t cell isolation kit, by STEMCELL (1 mentions) Iscove modified dulbecco media (imdm), by Merck Group (1 mentions) Anti cd28, by Merck Group (1 mentions) Rpmi media, by Merck Group (1 mentions) Anti cd3 antibody, by Merck Group (1 mentions) Anti hamster antibody, by MP Biomedicals (1 mentions) Macs gmp expact treg kit, by Miltenyi Biotec (1 mentions) Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions) Lymphocyte separation medium, by Corning (1 mentions) Il 27, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions) Clone cd28, by BioLegend (1 mentions)

Close spelling variants of this product

Isolation kits (7 citations) Cell isolation kits (4 citations)

11 protocols using human cd4 t cell isolation kit

CD4+ T Cell Activation Assay

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CD4+ T cells were isolated by negative selection from HC PBMCs (n=6) by a Human CD4+ T cell isolation kit following the instructions of the manufacturer (Stemcell). The T cells were subsequently stimulated with anti-CD3, anti-CD28 (BD Pharmingen) and IL-2 (Sigma-Aldrich) as previously described (49 (link)). The activated CD4+ T cells were used in two set-ups A. cultured with or without rhGal-9 (400ng/ml), rhGal-3 (400ng/ml) (R&D Systems, USA) analysis by ImageStream. B. Cells were cultured with, or without, 20% plasma from HCs (n=5) for 30 min. For the intracellular CD4 T cell analyses, cells were cultured under serum free conditions and activated with anti-CD3, anti-CD28 (BD Pharmingen) for 16 hours. Cells were then washed and media was added containing 20% HC plasma and Brefeldin A (10 μg/ml) for 30 min before cells were cultured on either a laminin coated (2 ug/ml) or uncoated surface for 30 min. Hereafter the CD4+ T cells were stimulated with rh4-1BBL (400ng/ml) for 4 hours. The samples were directly processed for further NanoSight, Flow cytometry or ImageStream analyses.

Nielsen M.A., Juul-Madsen K., Stegmayr J., Gao C., Mehta A.Y., Greisen S.R., Kragstrup T.W., Hvid M., Vorup-Jensen T., Cummings R.D., Leffler H, & Deleuran B.W. (2022). Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB. Frontiers in Immunology, 13, 915890.

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Treg Cell Isolation and Activation Assay

Cited 2 times

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CD25⁺ Treg cells were isolated from the leukapheresis product of end stage renal patients as previously described [16 (link),17 (link),18 (link)]. Briefly, the leukapheresis product was conjugated for 10 min at 4 °C with the CliniMACS CD25 MicroBeads reagent (Miltenyi Biotec). CD25⁺ cells were isolated in the Clini MACS Plus system (Miltenyi Biotec, software program Enrichment 3.2), following the manufacturer’s instructions. Treg cells were identified as CD4⁺/CD25⁺/FOXP3⁺/CD127⁻ and isolated with a purity above 85% throughout the study. Tconv were sorted from PBMCs with the human CD4⁺ T cell isolation kit (StemCell Technologies, Cambridge, MA, USA). For initial stimulation of cells, Treg and/or Tconv cells were placed in culture at a concentration of 10⁶ cells/mL in Treg cell culture medium: TexMACS GMP medium (Miltenyi) supplemented with recombinant human Interleukin-2 (IL2, MACS GMP, Miltenyi, at 500 IU/mL) and MACS GMP ExpAct Treg (beads conjugated to CD28, Anti-Biotin, and CD3-Biotin monoclonal antibodies, Miltenyi) at a bead-to-cell ratio of 2:1. Cells were exposed to treatment conditions of vehicle control (DMSO 0.1%), RAPA (100 nM) or AZD8055 (20 nM) for 60 min prior addition of cell activation supplements. Additionally, half of cell culture media was routinely replaced every three days.

Gedaly R., Orozco G., Ancheta A.P., Donoho M., Desai S.N., Chapelin F., Khurana A., Lewis L.J., Zhang C, & Marti F. (2023). Metabolic Disruption Induced by mTOR Signaling Pathway Inhibition in Regulatory T-Cell Expansion for Clinical Application. Cells, 12(16), 2066.

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Induction of Regulatory T Cells

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Naïve CD4⁺ T cells were purified from splenocytes using mouse CD4⁺ T cell isolation kit (Stemcell Technologies), and further sorted by FACS Aria II (BD Biosciences). 24-well plates were coated with 40 μg/ml anti-hamster antibody (MP Biomedical) at 37°C for 4 hours. For iTreg differentiation, naïve CD4⁺ T cells were cultured in T cell media IMDM (Sigma-Aldrich) supplemented with soluble 0.25 μg/ml anti-mouse CD3 (145–2C11), 1 μg/ml anti-mouse CD28 (37.51), 2 μg/ml anti-mouse IL-4 (11B11), 2 μg/ml anti-mouse IFN-γ (XMG1.2) and 5 ng/ml TGF-β, with (Th17) or without (iTreg) 20 ng/ml IL-6, for 3.5 days. For human cells, naïve T cells were enriched using the human CD4⁺ T cell isolation kit (Stemcell) from human PBMC cells and further sorted (CD3⁺CD4⁺CD45RA⁺CD25⁻). For iTreg differentiation, the sorted naïve human CD4⁺ T cells were seeded into 48-well plates precoated with 1 μg/mL anti-CD3 antibody (clone HIT3a), and cultured in RPMI media (Sigma-Aldrich) supplemented with IL-2 (10 ng/ml), soluble anti-CD28 (1 μg/ml; clone CD28.2) and 5 ng/ml TGF-β for 6 days. In some experiments, FICZ was added at a concentration of 200 nM as indicated in the text.

Xiong L., Dean J.W., Fu Z., Oliff K.N., Bostick J.W., Ye J., Chen Z.E., Mühlbauer M, & Zhou L. (2020). Ahr-Foxp3-RORγt Axis Controls Gut Homing of CD4+ T Cells by Regulating GPR15. Science immunology, 5(48), eaaz7277.

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Isolating and Culturing CD25+ Treg Cells

Cited 1 time

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CD25⁺ Treg cells were isolated from leukapheresis product of healthy volunteers using the CliniMACS Plus system (Miltenyi) as described^{23 (link)}. Treg cells were identified as CD4⁺/CD25⁺/FoxP3⁺/CD127⁻ and isolated with a purity above 85% throughout the study. Conventional, non Treg-Tcells (Tconv) were sorted from PBMCs with the human CD4⁺ T cell isolation kit (StemCell Technologies, Cambridge, MA). For initial stimulation of cells, Treg and/or Tconv cells were placed in culture at a concentration of 10⁶ cells/mL in Treg cell culture medium: TexMACS GMP medium (Miltenyi) supplemented with MACS GMP ExpAct Treg kit (Miltenyi) at a bead-to-cell ratio of 2:1 and recombinant human IL2 (rhIL-2 MACS GMP, Miltenyi, at 500 IU/mL). Rapa (100 nM), FH535 (1 µM) or Y3 (1 µM) were added where indicated. To assess the sequential effects of treatment with NABs and then Rapa (abbreviated as FH535 → Rapa or Y3 → Rapa), Rapa was added to the cells after initial 48-h in culture with FH535 or Y3 alone.

Gedaly R., Cornea V., Turcios L., Edmisson J.S., Harris D.D., Watt D.S., Chapelin F., Khurana A., Mei X., Liu C., Taylor I., Gonzalez-Valdivieso J., Mitchel H., Ruffing A., Chishti A., Orozco G., Zwischenberger J., Evers B.M, & Marti F. (2022). Anti-neoplastic sulfonamides alter the metabolic homeostasis and disrupt the suppressor activity of regulatory T cells. Scientific Reports, 12, 19112.

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Induction of Effector CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Lymphocyte Separation Medium (Corning). Total CD4⁺ T cells were isolated from PBMCs using a human CD4⁺ T cell isolation kit (STEMCELL Technologies, # 17952). To induce effector T cells, purified CD4⁺ T cells were stimulated for 72h using anti-CD3/CD28 beads (Life Technologies, # 11132D) at a ratio of 1 bead per 2 cells.

Li Y., Shen Y., Jin K., Wen Z., Cao W., Wu B., Wen R., Tian L., Berry G.J., Goronzy J.J, & Weyand C.M. (2019). The DNA repair nuclease MRE11A functions as a mitochondrial protector and prevents T cell pyroptosis and tissue inflammation. Cell metabolism, 30(3), 477-492.e6.

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Isolation and Activation of CD4+ T Cells

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PBMCs were isolated from healthy volunteers. CD4⁺ T cells were isolated using a human CD4⁺ T cell isolation kit (STEMCELL Technologies), according to manufacturer’s guidelines. CD4⁺ T cells (numbering 1 × 10⁶) were then cultured with soluble αCD28 (5 μg/mL, clone CD28.2, BioLegend) and plate bound αCD3ε (wells coated with 1 μg/mL for 4 hours at 37°C, 5% CO₂, clone UCHT1, BioLegend), supplemented with 50 IU/mL IL-2 (Miltenyi Biotech), 10 ng/mL IL-12 (BioLegend), 100 ng/mL IL-27 (Thermo Fisher Scientific) cell polarizing cytokines in a 48-well plate (for a final volume of 500 μL). After 3 days, cell culture supernatants were collected and stored at –20°C, and cells were assessed by flow cytometry.

Edwards C.L., Ng S.S., de Labastida Rivera F., Corvino D., Engel J.A., Montes de Oca M., Bukali L., Frame T.C., Bunn P.T., Chauhan S.B., Singh S.S., Wang Y., Na J., Amante F.H., Loughland J.R., Soon M.S., Waddell N., Mukhopadhay P., Koufariotis L.T., Johnston R.L., Lee J.S., Kuns R., Zhang P., Boyle M.J., Hill G.R., McCarthy J.S., Kumar R, & Engwerda C.R. (2023). IL-10-producing Th1 cells possess a distinct molecular signature in malaria. The Journal of Clinical Investigation, 133(1), e153733.

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Tregs Suppress CD4+ T Cell Proliferation

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Human peripheral blood mononuclear cells (PBMC) were isolated from normal healthy donor blood (New York Blood Center) with Ficoll (GE HealthCare, 17-1440-03) according to manufacturer’s instructions. Human CD4⁺ T cells were isolated from PBMC with human CD4⁺ T cell isolation kit (StemCell Technologies, 17952). Tregs were induced with human Tregs differentiation kit (R&D Systems, CDK006) according to manufacturer’s instructions. Autologous CD4⁺ T cells were label with carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, C34554). Tregs and autologous CD4⁺T cells were co-cultured with human T-activator CD3/CD28 Dynabeads (Gibco,11131D) for 4 days in the presence of AN3025 or human IgG₁, κ (BioxCell, BE0297). Proliferation of CD4⁺ T effector cells was evaluated with CFSE^lo population by flowcytometry (Sony, SA3800). Interferon gamma (IFNγ) production in the supernatant was detected by ELISA kit (R&D Systems, DY285B).

Chen Y., Jia M., Wang S., Xu S, & He N. (2022). Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity. Frontiers in Immunology, 13, 835690.

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Isolation and Activation of Primary Human CD4+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation from healthy anonymous blood donors. CD4+ T cells were negatively selected using magnetic beads as per manufacturer’s instructions (Human CD4+ T Cell Isolation Kit, Stemcell Technologies, Vancouver, BC, Canada; Cat.# 17952). CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 I.U. Penicillin, 100 μg/mL Streptomycin, 2 mM L-glutamine, and 30 Units/mL of rIL-2 (Peprotech, Rocky Hill, NJ, USA; Cat.# 200-02). Cells were stimulated with Human T-Activator CD3/CD28 Beads (Thermo Fisher Scientific, Waltham, MA, USA; Cat.#11132D) following manufacturer’s instructions and expanded in the presence of 30 Units/mL of rIL-2. Cells were infected by spinoculation at 1200× g for 1.5 h in multi-well plates. Experiments with primary T-cells were repeated with cells from eight different donors. CD14+ monocytes were isolated using magnetic beads as per manufacturer’s instructions (EasySep Human Monocyte Isolation Kit, Stemcell Technologies, Vancouver, BC, Canada; Cat.# 19359). Primary macrophages were further differentiated from the purified monocytes with recombinant human GM-CSF (10 ng/mL).

Langer S., Yin X., Diaz A., Portillo A.J., Gordon D.E., Rogers U.H., Marlett J.M., Krogan N.J., Young J.A., Pache L, & Chanda S.K. (2020). The E3 Ubiquitin-Protein Ligase Cullin 3 Regulates HIV-1 Transcription. Cells, 9(9), 2010.

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Isolation and Expansion of Human CD4+ T Cells

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Approximately 10 ml of human peripheral blood was obtained by venipuncture, rested in EDTA-K2 anticoagulation tubes, mixed with an equal volume of PBS, and slowly layered onto Lymphoprep (STEMCELL Technologies, Vancouver, BC, Canada, #07801) in SepMate tubes (STEMCELL Technologies, #86,450). CD4⁺ T cells were isolated from PBMCs using the human CD4⁺ T cell isolation kit (STEMCELL Technologies, # 17,952). To induce effector T cells, purified CD4⁺ T cells were cultured in ImmunoCul-XF T Cell Expansion Medium (STEMCELL Technologies, # 10,981) and supplemented with 25μL/mL human CD3/CD28 T Cell Activator (STEMCELL Technologies, # 10,971) and 10 ng/mL human recombinant IL-2 (STEMCELL Technologies, # 78,036).

Zhang J., Cai H., Sun W., Wu W., Nan Y., Ni Y., Wu X., Chen M., Xu H, & Wang Y. (2024). Endoplasmic reticulum aminopeptidase 2 regulates CD4+ T cells pyroptosis in rheumatoid arthritis. Arthritis Research & Therapy, 26, 36.

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Isolation of Lymphocyte Subsets from PBMC

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Lymphocyte subsets were purified from freshly prepared PBMC using EasySep (StemCell Technologies) negative magnetic selection kits: Human CD4⁺ T Cell Isolation Kit (StemCell Technologies, cat no. 17952), Human CD8⁺ T Cell Enrichment Kit (StemCell Technologies, cat no. 19053), Human NK Cell Enrichment Kit (StemCell Technologies, cat no. 19055) and Human Pan-B Cell Enrichment Kit (StemCell Technologies, cat no. 19554), per manufacturer’s instructions. After purification, 2 million (Pan B) or 3 million (CD4⁺ T, CD8⁺ T, and NK) isolated cells were lysed in Buffer RLT Plus (RNeasy Plus Mini Kit, Qiagen, cat no. 74134), per the manufacturer’s protocol, and lysates were kept at 4°C until RNA extraction. The remaining isolated cells were used for flow cytometric staining of markers to assess cell viability and purity as described below.

Woolley C.R., Chariker J.H., Rouchka E.C., Ford E.E., Hudson E.A., Waigel S.J., Smith M.L, & Mitchell T.C. (2022). Reference long-read isoform-aware transcriptomes of 4 human peripheral blood lymphocyte subsets. G3: Genes|Genomes|Genetics, 12(11), jkac253.

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