Cd4 cell isolation kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The CD4+ cell isolation kit is a laboratory tool used to separate and isolate CD4+ T cells from a sample. It provides a simple and effective method to obtain a purified population of CD4+ cells for further analysis or experimentation.

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Lab products found in correlation

Cd4 cd25 cd127dim regulatory t cell isolation kit 2, by Miltenyi Biotec (2 mentions) Ficoll hypaque, by Merck Group (2 mentions) Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Pe conjugated anti cd25, by BD (1 mentions) Ack lysis buffer, by Thermo Fisher Scientific (1 mentions) Ls column, by Miltenyi Biotec (1 mentions) Anti pe microbeads, by Miltenyi Biotec (1 mentions) Lenti x p24 rapid titer kit, by Takara Bio (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Anti pe beads, by Miltenyi Biotec (1 mentions) Macs ld column, by Miltenyi Biotec (1 mentions) Macs ls column, by Miltenyi Biotec (1 mentions) Lymphoprep density gradient medium, by STEMCELL (1 mentions) Dynabeads regulatory cd4 cd25 t cell kit, by Thermo Fisher Scientific (1 mentions) Histopaque, by Merck Group (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Human CD4+ T cells isolation kit CD4+CD25+CD127dim/– Regulatory T Cells Isolation Kit II CD4+CD25+Treg cells isolation kit Mouse Naïve CD4+ T Cells Isolation Kit CD4+ T cells isolation kit II CD4+ T cells isolation kit Naive CD4 T cells isolation kit Naïve CD4+T cells isolation kit CD4+CD25+ Regulatory T Cells Isolation Kit Cell isolation kits

10 protocols using cd4 cell isolation kit

PBMC Isolation and T Cell Purification

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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh, whole blood by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO, USA). CD4+ T cells and CD4+CD25+CD127dim/- Tregs were purified from PBMC using a CD4+ Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany) and CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II (Miltenyi) according to the manufacturer’s instruction, respectively.

Sanz-Rubio D., Sanz A., Varona L., Bolea R., Forner M., Gil A.V., Cubero P., Marin-Oto M., Martin-Burriel I, & Marin J.M. (2020). Forkhead Box P3 Methylation and Expression in Men with Obstructive Sleep Apnea. International Journal of Molecular Sciences, 21(6), 2233.

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Isolation and Activation of Murine Macrophages and T Cells

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Male wild-type (WT) mice (HFK Bioscience, Beijing, China) on the C57BL/6 background and aged 10-12 weeks were used in this study. Bone marrow-derived macrophages (Møs) were prepared as described previously [17 (link), 18 (link)]. The isolated macrophages were plated in complete DMEM supplemented with murine macrophage colony-stimulating factor (50 ng/ml) and cultured to allow macrophage differentiation. Spleens isolated from WT mice were mashed, and the splenocyte suspension was purified using a CD4+ cell isolation kit (Miltenyi Biotec, Auburn, CA). Then, the isolated T lymphocytes (TCs) were cultivated in complete RPMI 1640 medium and activated by treatment with anti-CD3 and anti-CD28 antibodies.

Hu H., Zhang G., Hu H., Liu W., Liu J., Xin S., Zhao X., Han L., Duan L., Huang X, & Chang C. (2019). Interleukin-18 Expression Increases in the Aorta and Plasma of Patients with Acute Aortic Dissection. Mediators of Inflammation, 2019, 8691294.

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Generation of Murine Regulatory T Cells

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A sterile single cell suspension was prepared from spleens and lymph nodes pooled from four young mice. Erythrocytes were lysed using ACK lysis buffer (Gibco by Life Technologies). CD4⁺ T cells were isolated by negative selection using Miltenyi’s CD4 cell isolation kit according to manufactures instructions. The CD4⁺ population was then stained with PE conjugated anti-CD25 (BD). After washing, the cells were then incubated with anti-PE microbeads (Miltenyi). CD25⁺ cells were removed by positive selection using the LS column (Miltenyi). Purity of the CD25⁻CD4⁺ T cells was confirmed by flow cytometry. The purified cells were suspended at 2 × 10⁶ cells/ml in X-Vivo15 serum free medium (Sartorius Stedim Biotech) and cultured in 24 well plates with Dynabeads^® Mouse T-Activator CD3/CD28 (Life Technologies) at a bead to cell ratio of 1:1. TGF-β1 (R&D Research Systems) was either added at 5000 pg/ml (recommended concentration for generation of regulatory T cells (Fantini et al.35 (link)) 100 pg/ml (concentration found in aged spleens), or 0 pg/ml (negative control). All conditions were plated in triplicate and cultured for 5 days at 37 °C 5%CO₂.

Lefebvre J.S., Masters A.R., Hopkins J.W, & Haynes L. (2016). Age-related impairment of humoral response to influenza is associated with changes in antigen specific T follicular helper cell responses. Scientific Reports, 6, 25051.

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Isolation and Activation of Primary CD4+ T Cells

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PBMCs were isolated from 50 to 100 ml of EDTA blood by gradient centrifugation (Ficoll-Paque Plus; GE Healthcare) and CD4⁺ T lymphocytes were enriched from the PBMC by negative magnetic separation (CD4⁺ Cell isolation Kit; Miltenyi Biotec). A fraction of these CD4⁺ T cells was stored following baseline (levels found prior to ex-vivo cell activation) cell-associated RNA and DNA measurements. Another fraction was resuspended in RPMI +10% fetal bovine serum (R10) at 2 × 10⁶ cells/ml. In most cases the cells were stimulated for 18 h with 50 ng/ml PMA (SIGMA) and 1 μM ionomycin (SIGMA). After activation, cells were washed with medium, counted and plated in cell culture wells with R10 + 30 U/ml IL-2. Co-cultures were then set up by adding activated uninfected donor CD4⁺ T cells to each well. In some cases the participant's cells were cultured by themselves i.e. no co-culture with feeder cells. In some experiments, 1 μM nevirapine (NVP) was added to the culture medium to prevent new rounds of viral replication. Cell cultures were monitored for viral production by measuring p24 antigen in the supernatant (Lenti-X p24 Rapid Titer Kit; Clontech). Cells were harvested when p24 was detectable in the supernatant and stored for analysis of cell -associated RNA and DNA as well as viral RNA in culture supernatants.

Vignoles M., Andrade V., Noguera M., Brander C., Mavian C., Salemi M., Paredes R., Sharkey M, & Stevenson M. (2021). Persistent HIV-1 transcription in CD4+ T cells from ART-suppressed individuals can originate from biologically competent proviruses. Journal of Virus Eradication, 7(3), 100053.

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Isolation of Naive T Cells from Spleen

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CD4⁺ cells from spleen were isolated by negative selection using CD4⁺ cell isolation kit and MACS LD column (Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer’s directions. Naïve T cells were further purified with PE-conjugated CD62L (MEL-14) antibodies, anti-PE beads, and MACS LS column (Miltenyi Biotec).

Kamiya T., Watanabe Y., Makino S., Kano H, & Tsuji N.M. (2016). Improvement of Intestinal Immune Cell Function by Lactic Acid Bacteria for Dairy Products. Microorganisms, 5(1), 1.

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Isolation of CD4+ T Cells and Tregs

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EDTA anticoagulant peripheral bloods were collected from each patients. PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO, USA). CD4⁺ T cells or CD4⁺CD25⁺CD127^dim/- Tregs were purified from PBMC using CD4⁺ Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany) or CD4⁺CD25⁺CD127^dim/- Regulatory T Cell Isolation Kit II (Miltenyi) according to manufacturer’s instruction, respectively. The purity of enriched cells was more than 95% by flow cytometry determination.

Yang L., Jia S., Shao X., Liu S., Zhang Q., Song J., Wang W, & Jin Z. (2019). Interleukin-35 modulates the balance between viral specific CD4+CD25+CD127dim/- regulatory T cells and T helper 17 cells in chronic hepatitis B virus infection. Virology Journal, 16, 48.

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Isolation of Splenic CD4+ CD25- T Cells

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Splenic CD4⁺ CD25⁻ T cells of all groups of mice were obtained from a double‐negative magnetic cell sorting (MACS). The Miltenyi CD4 cell isolation kit was used to label splenocytes with 100 μl of the biotinylated cocktail of antibodies (against CD8a, CD11b, CD11c, CD19, B220, CD49b, CD105, MHC‐class II and Ter‐119) specific for CD4⁻ cells diluted in 400 μl of RPMI 10% FBS for 15 min. at 4°C. Then, 200 μl of the anti‐biotin antibody conjugated with microbeads was added, and the cells were incubated for 10 min. at 4°C. After washing, the cells were applied on an LD separation column (Miltenyi, Teterow, Germany). CD4⁻ cells remained in the column because of the magnetic beads attached to their surface. By contrast, CD4⁺ cells flowed through the column and could be collected in a tube. The untouched CD4⁺ cells were further indirectly labelled with an anti‐CD25‐PE and anti‐PE conjugated with microbeads. Cells were flowed on an LS column, and CD4⁺ CD25⁻ cells were collected. The purity of CD4⁺ CD25⁻ T cells was more than 90% as identified by flow cytometry.

Xiao W., Yin M., Wu K., Lu G., Deng B., Zhang Y., Qian L., Jia X., Ding Y, & Gong W. (2016). High‐dose wogonin exacerbates DSS‐induced colitis by up‐regulating effector T cell function and inhibiting Treg cell. Journal of Cellular and Molecular Medicine, 21(2), 286-298.

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Isolation and Purification of CD4+ PBMCs

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Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of healthy volunteers. Blood was diluted at a 1:1 ratio with PBS then layered gently of Lymphoprep density gradient medium (STEMCELL, Cambridge, UK). Samples were centrifuged at 800G for 20 minutes to separate the layers then PBMC were harvested from the interface between the serum and lymphoprep layers. CD4+ cells were then isolated by negative selection using the CD4+ cell isolation kit (Miltenyi, Woking, UK). Purity was assessed by CD4 positivity using immunohistochemistry or flow cytometry (Figure S2).

Etherington R.E., Millar B.J., Innes B.A., Jones D.E., Kirby J.A, & Brain J.G. (2019). Bile acid receptor agonists in primary biliary cholangitis: Regulation of the cholangiocyte secretome and downstream T cell differentiation. FASEB bioAdvances, 1(5), 332-343.

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Isolation and Purification of CD4+ T Cells and Regulatory T Cells

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Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat by Histopaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. Isolated cells were washed by centrifugation in PBS (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) at 400× g at 4 °C for 10 min. CD4⁺ T cells were isolated using the CD4⁺ Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and the Treg cell population was isolated using the Dynabeads™ Regulatory CD4⁺CD25⁺ T Cell kit (Invitrogen). Both populations were isolated according to the detailed protocols provided by the manufacturers. The purity of isolated cells was evaluated by flow cytometry as described above.

Olkowska-Truchanowicz J., Sztokfisz-Ignasiak A., Zwierzchowska A., Janiuk I., Dąbrowski F., Korczak-Kowalska G., Barcz E., Bocian K, & Malejczyk J. (2021). Endometriotic Peritoneal Fluid Stimulates Recruitment of CD4+CD25highFOXP3+ Treg Cells. Journal of Clinical Medicine, 10(17), 3789.

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Comprehensive Immune Profiling of IciStem Patients

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This study included two IciStem patients. IciS-05 was included after signing a general waiver for the use of body material for future research, according to the regulations of the UMC Utrecht HSCT Biobank (HSCT study number 3440). IciS-11 was included after written informed consent was acquired. Ethical approval by the Institutional Medical Ethics Committee of the UMCU was obtained under protocol number NL53114.041.15. Before allo-HSCT, plasma, serum, PBMCs, and BM were obtained and a leukapheresis was performed. From the leukapheresis, 2–3 × 10⁸ PBMCs were sorted into T_N (CD3⁺CD4⁺CD45RO^-CCR7⁺CD27⁺Fas^-), T_SCM (CD3⁺CD4⁺CD45RO^-CCR7⁺CD27⁺Fas⁺), T_CM (CD3⁺CD4⁺CD45RO⁺CCR7⁺CD27⁺), T_TM (CD3⁺CD4⁺CD45RO⁺CCR7^-CD27⁺Fas⁺) and T_EM (CD3⁺CD4⁺CD45RO⁺CCR7^-CD27^-Fas⁺). CD4⁺ T-cells were obtained from baseline PBMCs and (CD4⁺-cell Isolation Kit, negative selection) (Miltenyi Biotec). At different time points after, allo-HSCT plasma and PBMCs were obtained. Post-mortem biopsies were obtained the day after death from terminal ileum, lung, liver, and spleen. From patient IciS-11, also brain biopsies were obtained and CD4⁺-cells were isolated from fresh ileum and a mediastinal lymph node.

Huyveneers L.E., Bruns A., Stam A., Ellerbroek P., de Jong D., Nagy N.A., Gumbs S.B., Tesselaar K., Bosman K., Salgado M., Hütter G., Brosens L.A., Kwon M., Diez Martin J., van der Meer J.T., de Kort T.M., Sáez-Cirión A., Schulze zur Wiesch J., Boelens J.J., Martinez-Picado J., Kuball J.H., Wensing A.M, & Nijhuis M. (2022). Autopsy Study Defines Composition and Dynamics of the HIV-1 Reservoir after Allogeneic Hematopoietic Stem Cell Transplantation with CCR5Δ32/Δ32 Donor Cells. Viruses, 14(9), 2069.

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