Cd4 positive selection kit

Manufactured by STEMCELL

Sourced in Canada

The CD4 positive selection kit is a laboratory tool used for the isolation and enrichment of CD4+ T cells from a heterogeneous cell population. It functions by selectively binding and separating CD4+ cells from the rest of the sample.

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EasySep mouse CD4+ T cell pre-enrichment and CD25 positive selection kits (2 citations)

6 protocols using cd4 positive selection kit

Isolation of Human NK and T Cells

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PBMCs were isolated by Ficoll-Paque (GE Healthcare, Chalfont St Giles, England) density gradient centrifugation. Human NK cells were purified either from whole blood using the RosetteSep Human NK cell enrichment Cocktail, or from PBMC using Human NK cell negative selection kit (StemCell Technologies, Vancouver, BC, Canada). Human Treg-depleted T cells were purified from PBMC using a human T cell enrichment kit and CD25 positive selection kit; CD4⁺ T cells were purified from PBMC using a human T cell enrichment kit and CD4 positive selection kit (StemCell Technologies, Vancouver, BC, Canada). The purity of isolated NK cells and T cells were confirmed to be above 90%.

Lin L., Ma C., Wei B., Aziz N., Rajalingam R., Yusung S., Erlich H.A., Trachtenberg E.A., Targan S.R., McGovern D.P., Heath J.R, & Braun J. (2014). Human Natural Killer Cells Licensed by KIR Genes Have an Altered Cytokine Program That Modifies CD4+ T Cell Function. Journal of immunology (Baltimore, Md. : 1950), 193(2), 940-949.

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Regulatory T Cell Suppression Assay

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T effector (Teff) cells (CD4⁺CD25^-CD45.1⁺) were isolated (>95% purity) from spleens and lymph nodes of naïve CD45.1 congenic BALB/c mice by magnetic bead separation using a CD4 positive selection kit (StemCell) and subsequent depletion of CD25⁺ cells (CD25⁺ positive selection kit; StemCell). Treg cells (CD4⁺CD25⁺CD45.2⁺) were purified (>95%) from spleens and lymph nodes of naïve BALB/c, SKG or Nod2^−/−SKG mice using EasySep™ Mouse CD4⁺CD25⁺ Regulatory T Cell Isolation Kit II (StemCell). Teff cells (1 × 10⁵/well) were pre-treated with 5μM of the cell proliferation dye Cell Trace Violet (ThermoFisher) and cultured with indicated numbers of Tregs, along with 2 × 10⁵ CD45.1+ autologous BALB/c APCs that had been irradiated (1000 rads). Cultures were stimulated with 0.5μg/ml plate bound anti-CD3 (145–2C11, eBiosciences). Proliferation of Teff cells was assessed 72h post stimulation by Cell Trace Violet dilution on the BD LSRFortessa™ (BD Biosciences) and analyzed using FlowJo software (FlowJo, LLC).

Napier R.J., Lee E.J., Vance E.E., Snow P., Samson K.A., Dawson C.E., Moran A.E., Stenzel P., Davey M.P., Sakaguchi S, & Rosenzweig H.L. (2018). Nod2-deficiency augments Th17-responses and exacerbates autoimmune arthritis. Journal of immunology (Baltimore, Md. : 1950), 201(7), 1889-1898.

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Isolation and Polarization of Mouse Dendritic Cells and Macrophages

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For bone marrow-derived dendritic cells (BMDCs) preparation, bone marrow from 6 to 8-week-old C57BL/6 mice was cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), recombinant mouse GM-CSF (50 ng/mL; Peprotech), and IL-4 (20 ng/mL; Peprotech). Half medium was replaced by fresh medium with GM-CSF and IL-4 at day 3, and used at day 6 as immature DCs. Immature DCs were stimulated with TLR agonists LPS (50 ng/mL; Sigma-Aldrich) or poly I:C (20 ng/mL; InvivoGen) for 8 h to generate mature DCs.
For bone marrow-derived macrophages (BMDMs) preparation, bone marrow from 6 to 8-week-old C57BL/6 mice was cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), recombinant mouse M-CSF (50 ng/mL; Peprotech). All supernate was replaced by fresh medium with M-CSF at day 3. BMDMs were then stimulated with LPS (50 ng/mL; Sigma-Aldrich) for M1 polarization, or IL-4 (20 ng/mL; Peprotech) for M2 polarization for 24 h at day 6.
CD11c⁺ DCs were isolated from the spleen using CD11c positive selection kit (STEMCELL Technologies), and CD4⁺ T cells were isolated from the lymph nodes using CD4 positive selection kit (STEMCELL Technologies).

Wang J., Wang J., Hong W., Zhang L., Song L., Shi Q., Shao Y., Hao G., Fang C., Qiu Y., Yang L., Yang Z., Wang J., Cao J., Yang B., He Q, & Weng Q. (2021). Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling. Nature Communications, 12, 6198.

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Generation of hiPS-CAR-T Cells from CD8+ T Cells

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The harvested DP T lymphocytes were transferred to lymphoid differentiation material-coated plates, and 10 ng/mL IL-2, 10 ng/mL IL-15, and 6.25 µL human CD3/CD28/CD2 T cell activator were added to each well for 7–14 days for maturation and differentiation of CD8⁺ T cells (Minagawa et al., 2018 (link); Hu et al., 2020 (link); Ji et al., 2022 (link); Wu et al., 2022 ; Jian et al., 2023 (link)). Except for mature T cells, most immature lymphocytes, including CD4⁺CD8⁺ DP T cells, died as a result of T cell receptor (TCR) rearrangement. CD3⁺ T cells were sorted out using the CD3 Positive Selection Kit II (stemcell#17851), followed by CD4⁻CD8⁺ T cells (hiPS-T) using the CD4 Positive Selection Kit (stemcell, #17852) and CD8 Positive Selection Kit (stem cell, #17853). From the CD3⁺ T cells obtained above, CD4⁻CD8⁺ T cells (hiPS-T) were isolated, purified, and set aside. The harvested hiPS-T cells were infected with CD19 CAR lentivirus (5 μg/mL polybrene + CAR lentivirus (MOI = 7.5)) (Figure 1B) for 24 h to obtain hiPS-CAR-T cells.

Chen Y., Huang P., Niu M., Tian C., Zhang T, & Peng Z. (2023). Regeneration of T cells from human-induced pluripotent stem cells for CAR-T cell medicated immunotherapy. Frontiers in Bioengineering and Biotechnology, 11, 1159507.

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Isolation and Transfer of NK Cells

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Transferred NK1.1⁺CD11c⁺ cells were sorted by FACSAria (BD Bioscience) or RoboSep (Stemcell Technology). Transferred NK1.1⁺ cells were purified by a combination of CD4-negative / NK1.1 and CD11c-positive bead selection (RoboSep, Stemcell Technologies) from cell suspension depleted of CD4 cells by CD4-positive-selection kit (Stemcell Technologies). 3-5×10⁶ cells were injected i.v. per mouse. Recipients were untouched WT mice.

Voynova E.N., Skinner J, & Bolland S. (2015). Expansion of an atypical NK cell subset in mouse models of SLE. Journal of immunology (Baltimore, Md. : 1950), 194(4), 1503-1513.

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CD4+ T Cell Expansion and CCR5 Editing

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CD4 cells were isolated from peripheral blood of the healthy donor using CD4 positive selection kit (Cat No: #17852, stem cell technologies) as per the manufacturer’s instructions and the purity was analyzed using CD3 and CD4 FACS antibodies. Isolated cells were cultured using human XF T cell expansion medium (Cat No: 10981, stem cell technologies) along with CD3/CD28/CD2 T cell activator cocktail (Cat No: 10970, Stem cell technologies). CCR5 gene editing was performed on day 4 of expansion.

Karuppusamy K.V., Demosthenes J.P., Venkatesan V., Christopher A.C., Babu P., Azhagiri M.K., Jacob A., Ramalingam V.V., Rangaraj S., Murugesan M.K., Marepally S.K., Varghese G.M., Srivastava A., Kannangai R, & Thangavel S. (2022). The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy. Frontiers in Immunology, 13, 792684.

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