cd4 t cell isolation kit, by Miltenyi Biotec - Product details

1

Dendritic Cell-Mediated OVA-Specific T Cell Activation

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BM-derived DCs (BMDCs) were prepared as follows. BM cells were flushed from femur and tibia of WT C57BL/6J mice and cultured in RPMI media (Sigma) containing 10% FSC and 1% Penicillin/Streptomycin (Gibco) supplemented with GM-CSF (5 ng/ml). Fresh media containing GM-CSF was added at day 3 and 5 of cultivation. After 7 days, BMDCs was pulsed with OVA cognate peptide 323-339 (irrelevant OVA 257–264 peptide was used as control) (InvivoGen) at a concentration of 1 μg/ml and co-cultivated with OVA-specific OT-II T cells and Tregs (10 000 BMDCs: 50 000 OT-II T cells: 50 000 Tregs). OT-II T cells were isolated from OT-II⁺Rag1^−/− mice as MACS-enriched CD4⁺ T cells (CD4⁺ T Cell Isolation Kit, Miltenyi biotec). CD4⁺ conventional T cells (Tconv) were used as a negative control. Tregs were isolated from LNs (pLN and mLN) of WT (MyD88^fl/fl) and MyD88^ΔTECs mice using subsequent Auto-MACS (Miltenyi biotec) procedure. CD4-enriched T cells (CD4⁺ T Cell Isolation Kit, Miltenyi biotec) were stained by anti-CD25 biotin conjugated antibody and CD4⁺CD25⁺ Tregs were isolated using Anti-Biotin MicroBeads (Miltenyi biotec). Tconv cells were prepared using Auto-MACS as CD4⁺CD25^– cells. After 3 days of co-cultivation, cells were stained with anti-Vβ5 and anti-Vα2 antibodies to distinguish OT-II⁺ T cells. Proliferation was measured by FACS using CPD670 staining.

Vobořil M., Brabec T., Dobeš J., Šplíchalová I., Březina J., Čepková A., Dobešová M., Aidarova A., Kubovčiak J., Tsyklauri O., Štěpánek O., Beneš V., Sedláček R., Klein L., Kolář M, & Filipp D. (2020). Toll-like receptor signaling in thymic epithelium controls monocyte-derived dendritic cell recruitment and Treg generation. Nature Communications, 11, 2361.

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CD4+ T Cell Isolation and Activation

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Following removal of dead cells, CD4⁺ T cells were isolated using negative magnetic bead selection with the CD4⁺ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications. This negative selection process depletes CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and CD235a and delivers untouched CD3⁺CD4⁺ T cells. Additionally, anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4⁺ T cell population was higher than 90% (see Figure 2A). Yield recovery of CD4⁺ T cells is shown in Supplementary Figure 1b. Following isolation, cells were analyzed by RT-PCR as described below, without in vitro stimulation.
CD4⁺ T cells from blood were isolated with the CD4⁺ T cell isolation kit (Miltenyi Biotec) following standard ficoll centrifugation, and activated in vitro with Phytohemagglutinin (PHA) (2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann- La Roche Inc)^{38 (link)} for 24h as described before^{8 (link)}.

Rodriguez-Garcia M., Barr F.D., Crist S.G., Fahey J.V, & Wira C.R. (2014). Phenotype and Susceptibility to HIV-infection of CD4+ Th17 Cells in the Human Female Reproductive Tract. Mucosal immunology, 7(6), 1375-1385.

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Adoptive Transfer and Pathogen Infection

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For LLO56, LLO118, and LLO118⁺Scn5a⁺Cd4-creErt2⁺ transfer experiments, CD4⁺ T cells were enriched from spleen by negative selection using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). Cells were then transferred iv into recipient mice, and 12-24 hours later the mice were either infected iv with 10⁷ cfu of actA-deficient Listeria monocytogenes (strain DPL1942^{48 (link)}) or immunized as indicated. NP-conjugated protein immunizations were performed by ip injection of mice with 100μg of the conjugated protein in equal parts adjuvant aluminum hydroxide gel, Alhydrogel (InvivoGen). For peptide immunizations, mice were injected ip with 10μM peptide in 200μl of a 1:1 PBS:Alhydrogel mixture. For the Nur77-high/low and CD5-high/low transfers, CD4⁺ T cells were first negatively selected from spleen using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec) and then stained for CD4, CD44, CD62L, CD25, and CD5. Naive CD4⁺ T cells (CD4⁺/CD44^lo/CD62L⁺/CD25⁻) in the top and bottom 25% of Nur77-GFP or CD5 expression were sorted using a BD FACSAriaII cytometer. 7.5-10x10⁶ cells were injected iv into recipient mice (transferred amounts varied across experiments, not within). One day later, recipient mice were infected ip with 2x10⁵ PFU Armstrong strain LCMV^{49 (link)}.

Bartleson J.M., Milam A.A., Donermeyer D.L., Horvath S., Xia Y., Egawa T, & Allen P.M. (2020). Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions. Nature immunology, 21(11), 1384-1396.

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Adoptive Transfer and Pathogen Infection

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For LLO56, LLO118, and LLO118⁺Scn5a⁺Cd4-creErt2⁺ transfer experiments, CD4⁺ T cells were enriched from spleen by negative selection using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). Cells were then transferred iv into recipient mice, and 12-24 hours later the mice were either infected iv with 10⁷ cfu of actA-deficient Listeria monocytogenes (strain DPL1942^{48 (link)}) or immunized as indicated. NP-conjugated protein immunizations were performed by ip injection of mice with 100μg of the conjugated protein in equal parts adjuvant aluminum hydroxide gel, Alhydrogel (InvivoGen). For peptide immunizations, mice were injected ip with 10μM peptide in 200μl of a 1:1 PBS:Alhydrogel mixture. For the Nur77-high/low and CD5-high/low transfers, CD4⁺ T cells were first negatively selected from spleen using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec) and then stained for CD4, CD44, CD62L, CD25, and CD5. Naive CD4⁺ T cells (CD4⁺/CD44^lo/CD62L⁺/CD25⁻) in the top and bottom 25% of Nur77-GFP or CD5 expression were sorted using a BD FACSAriaII cytometer. 7.5-10x10⁶ cells were injected iv into recipient mice (transferred amounts varied across experiments, not within). One day later, recipient mice were infected ip with 2x10⁵ PFU Armstrong strain LCMV^{49 (link)}.

Bartleson J.M., Milam A.A., Donermeyer D.L., Horvath S., Xia Y., Egawa T, & Allen P.M. (2020). Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions. Nature immunology, 21(11), 1384-1396.

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5

Isolation and Characterization of CD4+ T Cell Subsets

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Peripheral blood samples were collected into EDTA vacutainer tubes (Greiner Bio-One). PBMCs were isolated from the blood of healthy volunteers, RA, and PsA patients by density gradient centrifugation over Histopaque-1077 (Sigma-Aldrich). For quantitative real-time PCR analysis, the CD4⁺ T cells were negatively isolated from PBMCs with a magnetic-activated cell sorting CD4⁺ T Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. The CD45RO⁻ (negative fractions) and CD45RO⁺ (positive fractions) cells were separated also with a CD45RO Microbeads (Miltenyi Biotec). For flow cytometric analysis of chemokine receptors, the CD4⁺CD45RO⁻ cells were negatively isolated from PBMCs with naive CD4⁺ T Cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of the separated cells was at least 95% in all cases, as determined by flow cytometry.

Baricza E., Marton N., Királyhidi P., Kovács O.T., Kovácsné Székely I., Lajkó E., Kőhidai L., Rojkovich B., Érsek B., Buzás E.I, & Nagy G. (2018). Distinct In Vitro T-Helper 17 Differentiation Capacity of Peripheral Naive T Cells in Rheumatoid and Psoriatic Arthritis. Frontiers in Immunology, 9, 606.

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CD4+ T Cell Isolation and Activation

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Following removal of dead cells, CD4⁺ T cells were isolated using negative magnetic bead selection with the CD4⁺ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications. This negative selection process depletes CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and CD235a and delivers untouched CD3⁺CD4⁺ T cells. Additionally, anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4⁺ T cell population was higher than 90% (see Figure 2A). Yield recovery of CD4⁺ T cells is shown in Supplementary Figure 1b. Following isolation, cells were analyzed by RT-PCR as described below, without in vitro stimulation.
CD4⁺ T cells from blood were isolated with the CD4⁺ T cell isolation kit (Miltenyi Biotec) following standard ficoll centrifugation, and activated in vitro with Phytohemagglutinin (PHA) (2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml) (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann- La Roche Inc)^{38 (link)} for 24h as described before^{8 (link)}.

Rodriguez-Garcia M., Barr F.D., Crist S.G., Fahey J.V, & Wira C.R. (2014). Phenotype and Susceptibility to HIV-infection of CD4+ Th17 Cells in the Human Female Reproductive Tract. Mucosal immunology, 7(6), 1375-1385.

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7

Isolation of PBMCs from Atopic Dermatitis Patients

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Residual blood samples from platelet (PLT) apheresis disposables used for routine PLT collection of regular anonymous healthy donors (buffy coats) served as source material for the isolation of human peripheral blood mononuclear cells (PBMCs). Venous blood samples were taken from healthy control persons (Caucasian, n = 10, 4 females and 6 males mean age 29.3 years) and from patients with moderate-to-severe extrinsic atopic dermatitis (AD) (Caucasian, n = 8, 1 female and 7 males mean age 33,25 years). Mean SCORAD was 35.0 ranging from 13.6 to 62.4. Three AD patients received dupilumab as systemic treatment, the others received no systemic treatment. AD patients and healthy control persons were recruited from the Department of Dermatology and Allergy, Hannover Medical School, Hannover, Germany. AD was diagnosed according to the criteria of Hanifin and Rajka [20 ].
PBMCs were separated by density gradient centrifugation on Pancoll human (PAN-Biotech, Aidenbach, Germany) and afterwards naïve or total CD4⁺ T cells were isolated using respective CD4⁺ T cell Isolation Kits (Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Dawodu D., Sand S., Nikolouli E., Werfel T, & Mommert S. (2023). The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4+ T cells. Inflammation Research, 72(8), 1525-1538.

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CD4+ T Cell Isolation and Characterization

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In the AAD model, lymph node cells (LNC) from HLN and ILN were processed using CD4⁺ T cell isolation kits (Miltenyi Biotec) to separate CD4⁺ from CD4⁻ cell populations. LNC were also dissected from draining popliteal lymph nodes after s.c. immunization with MOG₃₅₋₋₅₅peptide, an autoantigen recognized by T cells in EAE and MS (Preller et al., 2007 (link)). Concanavalin A (Con A) activated mouse splenocytes as a source of T cell blasts were prepared and cultured as described (Dong et al., 2006 (link); Vang et al., 2010 (link)). Cells were either immediately frozen in appropriate reagents for subsequent qRT-PCR or Western immunoblot analyses or used in proliferation assays as described (Vang et al., 2013 (link)).

Vang A.G., Basole C., Dong H., Nguyen R.K., Housley W., Guernsey L., Adami A.J., Thrall R.S., Clark R.B., Epstein P.M, & Brocke S. (2016). Differential Expression and Function of PDE8 and PDE4 in Effector T cells: Implications for PDE8 as a Drug Target in Inflammation. Frontiers in Pharmacology, 7, 259.

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9

Isolation of Dendritic Cells and T Cells

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DC2.4 and THP-1 cell lines were gifts from K. Rock (University of Massachusetts Medical School, Worcester, MA) and P. Cresswell of (Yale University, New Haven, CT), respectively. To derive DCs from the bone marrow, nonadherent marrow cells from the tibia and femur bones were cultured in the presence of 3 ng/ml GM-CSF and IL-4 for 6 d. Human peripheral blood was collected from healthy volunteers according to approved guidelines of University of Calgary, and T reg cells from these human blood samples were isolated using the human CD4⁺CD127^lowCD49D⁻CD25⁺ regulatory T cell magnetic purification kit purchased from StemCell Technologies, Inc. Murine CD4⁺CD25⁺ T reg cells and CD4⁺CD25⁻ T conv cells were isolated from spleens using magnetic purification kits from either Miltenyi Biotec or StemCell Technologies, Inc. OT-II T cells were isolated from OT-II splenocytes by Miltenyi Biotec CD4 T cell isolation kits and sometimes sorted by FACS with an anti-TCR Vα2 antibody.

Chen J., Ganguly A., Mucsi A.D., Meng J., Yan J., Detampel P., Munro F., Zhang Z., Wu M., Hari A., Stenner M.D., Zheng W., Kubes P., Xia T., Amrein M.W., Qi H, & Shi Y. (2017). Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy. The Journal of Experimental Medicine, 214(2), 327-338.

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10

OVA-specific T cell activation

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C57BL/6 J mice were injected with 100 ug OVA and 1 mg Imject™ Alum Adjuvant at day 1 to induce OVA-specific T-cells. And then injecting with alum-OVA again at day 5. The mice were sacrificed at day 14. T cells in lymph nodes and spleen were isolated utilizing CD4⁺ T Cell isolation Kits (Miltenyi Biotec). The GCA pretreated or untreated 3T3-L1 cells were cocultured with CD4⁺ T Cells for 48 h in the presence/absence of 200 μg/ml OVA. Then, the cell supernatants were analyzed by ELISA and T cells were collected for flow cytometry analyses.

Su T., He Y., Huang Y., Ye M., Guo Q., Xiao Y., Cai G., Chen L., Li C., Zhou H, & Luo X. (2024). Myeloid-derived grancalcin instigates obesity-induced insulin resistance and metabolic inflammation in male mice. Nature Communications, 15, 97.

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Cd4 t cell isolation kit

Lab products found in correlation

605 protocols using cd4 t cell isolation kit

Dendritic Cell-Mediated OVA-Specific T Cell Activation

CD4+ T Cell Isolation and Activation

Adoptive Transfer and Pathogen Infection

Adoptive Transfer and Pathogen Infection

Isolation and Characterization of CD4+ T Cell Subsets

CD4+ T Cell Isolation and Activation

Isolation of PBMCs from Atopic Dermatitis Patients

CD4+ T Cell Isolation and Characterization

Isolation of Dendritic Cells and T Cells

OVA-specific T cell activation

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