Easysep mouse cd4 positive selection kit 2

Manufactured by STEMCELL

Sourced in Canada

The EasySep™ Mouse CD4 Positive Selection Kit II is a tool for isolating mouse CD4+ T cells from a single-cell suspension. It uses magnetic particles to select the desired cell population, resulting in a highly pure sample.

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Lab products found in correlation

Easysep mouse pan dc enrichment kit 2, by STEMCELL (1 mentions) Cd68 pe clone fa 11, by BioLegend (1 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (1 mentions) Easysep mouse nk cell isolation kit, by STEMCELL (1 mentions) Easysep pe positive selection kit, by STEMCELL (1 mentions) Mouse il 12, by Thermo Fisher Scientific (1 mentions) Soluble anti cd3, by BioXCell (1 mentions) Mouse il 4, by BioLegend (1 mentions) Anti ifn γ, by BioXCell (1 mentions) Anti il 12, by BioXCell (1 mentions) Mouse il 6, by BioLegend (1 mentions) Anti il 4, by BioXCell (1 mentions) Anti cd28, by BioXCell (1 mentions) Human tgf β, by Thermo Fisher Scientific (1 mentions) Click s medium, by Irvine Scientific (1 mentions) Rnalater, by Thermo Fisher Scientific (1 mentions) Anti cd4, by BioLegend (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Merck Group (1 mentions) Percoll, by Merck Group (1 mentions)

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EasySep kits (21 citations)

6 protocols using easysep mouse cd4 positive selection kit 2

Immune Cell Isolation from Murine Spleens

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Spleens from the tumor-bearing C57BL/6 mice or cryo-thermal treated mice were harvested and splenocytes were prepared using GentleMACS™ dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) and passed through a 40-μm nylon filter. CD4⁺ T cells were isolated by EasySep™ Mouse CD4 Positive Selection Kit II (StemCell Technologies, Vancouver, BC, Canada). CD8⁺ T cells were isolated by EasySep™ Mouse CD8⁺ T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada). Natural Killer (NK) cells were isolated by EasySep™ Mouse NK Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada). CD68⁺ macrophages were isolated by EasySepTM PE positive selection kit (StemCell Technologies, Vancouver, BC, Canada) and CD68-PE (clone FA-11, Biolegend, San Diego, CA, USA). DCs were isolated by EasySep™ Mouse Pan-DC Enrichment Kit II (StemCell Technologies, Vancouver, BC, Canada). Cells were all isolated according to the manufacturer’s instructions. Cells with a purity of >90% were used for experiments.

Peng P., Lou Y., Wang J., Wang S., Liu P, & Xu L.X. (2022). Th1-Dominant CD4+ T Cells Orchestrate Endogenous Systematic Antitumor Immune Memory After Cryo-Thermal Therapy. Frontiers in Immunology, 13, 944115.

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Isolation and Polarization of Mouse CD4+ T Cells

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CD4⁺ T cells were isolated from the spleen and lymph nodes using the EasySep Mouse CD4 Positive Selection Kit II (Stemcell Technologies). Naive (CD25⁻CD44^hiCD62L^lo) CD4⁺ T cells were sorted by flow cytometry from the bead purified CD4⁺ T cells. The naive CD4⁺ T cells were resuspended in Click’s medium (Irvine Scientific) at 1 million cells per ml, and then plated on day 0 in 24 well plates coated with goat anti-hamster IgG antibody (200 ng ml⁻¹; MP Biomedicals) with the addition of soluble anti-CD3 (1 μg ml⁻¹; 145-2C11) and anti-CD28 (1 μg ml⁻¹; 37.51) from Bio X Cell. Polarizing conditions for different T helper subsets are as following: T_H1: human IL-2 (100 U ml⁻¹; PeproTech), mouse IL-12 (20 ng ml⁻¹; PeproTech) and anti-IL-4 (5 μg ml⁻¹; Bio X Cell); T_H2: human IL-2 (100 U ml⁻¹; PeproTech), mouse IL-4 (20 ng ml⁻¹; Biolegend), anti-IFN-γ and anti-IL-12 (5 μg ml⁻¹; Bio X Cell); T_H17: mouse IL-6 (20 ng ml⁻¹; Biolegend), human TGF-β (2 ng ml⁻¹; PeproTech), anti-IFN-γ and anti-IL-12 (5 μg ml⁻¹; Bio X Cell).

Bapat S.P., Whitty C., Mowery C.T., Liang Y., Yoo A., Jiang Z., Peters M.C., Zhang L.J., Vogel I., Zhou C., Nguyen V.Q., Li Z., Chang C., Zhu W.S., Hastie A.T., He H., Ren X., Qiu W., Gayer S.G., Liu C., Choi E.J., Fassett M., Cohen J.N., Sturgill J.L., Alexander L.E., Suh J.M., Liddle C., Atkins A.R., Yu R.T., Downes M., Liu S., Nikolajczyk B.S., Lee I.K., Guttman-Yassky E., Ansel K.M., Woodruff P.G., Fahy J.V., Sheppard D., Gallo R.L., Ye C.J., Evans R.M., Zheng Y, & Marson A. (2022). Obesity alters pathology and treatment response in inflammatory disease. Nature, 604(7905), 337-342.

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Isolation and Purification of Mouse CD4+ T Cells

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Splenocytes were isolated from mice as described above and CD4⁺ T cells were sorted using the EasySep Mouse CD4-Positive Selection Kit II (Stem Cell Technologies) following the manufacturer’s instructions. Aliquots of sorted cells were stained with anti-CD4 (GK1.5 Biolegend) to assess the cell purity by flow cytometry and the remaining samples were then preserved in RNAlater (Invitrogen) and kept at 4°C for subsequent RNA isolation. The purity of sorted splenic CD4⁺ T cells was confirmed to be ~90% by flow cytometry.

Abushahba M.F., Dadelahi A.S., Ponzilacqua-Silva B., Moley C.R, & Skyberg J.A. (2024). Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection. mSphere, 9(3), e00750-23.

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Isolation of Intestinal CD4+ T-cells

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The lumenal content was removed, and the sectioned intestine was washed with ice-cold PBS. The tissue was then cutted into 1-2 mm pieces and digested with 2 mg/ml Collagenase D (Cat#: 11088858001, Roche) and 2 mg/ml DNase I (Cat#: 10104159001, Sigma-Aldrich) in RPMI-1640 medium (Gibco) at 37°C for 40 min under slow rotation. The supernatants were filtered by 70 µm mesh. The collected cells were resuspended in 30% Percoll (Sigma) and carefully overlaid onto 70% Percoll. After centrifugation, the cell layers located between the two Percoll layers were collected. CD4⁺ T-cells were isolated using EasySep™ Mouse CD4 Positive Selection Kit II (Cat#: 18952, STEMCELL Technologies, Canada).

Song S., Lou Y., Mao Y., Wen X., Fan M., He Z., Shen Y., Wen C, & Shao T. (2022). Alteration of Gut Microbiome and Correlated Amino Acid Metabolism Contribute to Hyperuricemia and Th17-Driven Inflammation in Uox-KO Mice. Frontiers in Immunology, 13, 804306.

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Isolation of Murine Splenocyte Subsets

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Splenocytes were isolated from female wildtype BALB/c mice spleens by using a cell strainer, and subsequent collection of the cells in phosphate buffered saline (PBS). Residual red blood cells were removed through addition of Ammonium-Chloride-Potassium lysing buffer, before the remaining splenocytes were resuspended in RPMI 1640 medium containing 10% FCS, L-glutamine, pyruvate, the antibiotics penicillin and streptomycin and 2μM 2-Betamercaptoethanol (Sigma-Aldrich).
Several splenocyte samples were further processed to isolate CD4⁺ and CD8⁺ cells using the following kits from Stemcell Technologies: EasySep™ Mouse CD4 Positive Selection Kit II (Cat. No 18952RF) and EasySep™ Mouse CD8⁺ T Cell Isolation Kit (Cat No 19853RF). The cells were isolated according to the manufacturer’s protocols.

Sparrow E.L., James S., Hussain K., Beers S.A., Cragg M.S, & Bogdanov Y.D. (2021). Activation of GABA(A) receptors inhibits T cell proliferation. PLoS ONE, 16(5), e0251632.

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Isolation of 2C-like mouse ESCs

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The 2C-GFP reporter construct (Ishiuchi et al. 2015) was modified to insert a T2A cleavage element followed by the extracellular portion of mouse CD4 (a.a.1-427) immediately downstream of GFP, so that activation of MERVL in the 2C-like state labels cells doubly positive for GFP and CD4. ESCs are negative for CD4 expression, enabling rapid purification of endogenous 2C-like cells via selection for CD4+ surface expression. E14 ESCs were nucleofected with 4ug linearized 2C-GFP plasmid and plated at low density in 10cm 2 plates then selected with 250 µg/mLG418 (Mirus) for 8 days. Individual colonies were picked and expanded, with a single colony that showed high specific expression of GFP expanded and used for subsequent validations and experiments. For 2C-GFP/CD4 isolation, cells were trypsinised, washed, and resuspended in FACS buffer (PBS, 3% FBS, 1mM EDTA) then either isolated by MACS, using CD4 (L3T4) microbeads (Miltenyi Biotec), or with the EasySep mouse CD4 Positive selection kit II (StemCell), according to the manufacturer's protocols in each case. Apart from Figure S1, all purification experiments were performed with the EasySep kit. Flow-through cells were collected as the 2C-negative population. For flow cytometry analysis, ESCs were pelleted and resuspended in FACS buffer containing 1:8000 Sytox Blue (Thermo Fisher Scientific) to enable exclusion of dead cells.

Xie S., Leeke B., Whidling C., Wagner R.T., Garcia-Llagostera F., Chammas P., Cheung N.T., Dormann D., McManus M.T., & Percharde M. (2021). Nucleolar-based Dux repression is essential for 2-cell stage exit.

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