Cd4 dynabeads

Manufactured by Thermo Fisher Scientific

Sourced in Norway

Thermo Fisher Scientific's CD4 Dynabeads are magnetic beads coated with antibodies specific to the CD4 surface marker. They are designed for the isolation and enrichment of CD4+ T cells from various sample types, such as whole blood or cell suspensions. The beads can be used in combination with appropriate cell separation and downstream analysis techniques.

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14 protocols using cd4 dynabeads

Detecting IFN-γ-producing CD8 T Cells

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In order to detect only IFN-γ-producing CD8 T cells, ELISPOT assays were carried out with PBMCs or lymph node mononuclear cells (LNMC) after negative selection of CD4 T-lymphocyte populations fractionated by magnetic bead separation (CD4 Dynabeads; Dynal, Oslo, Norway) as previously described [20 (link)]. Negatively selected CD4 T cells were >90% CD8 T cells. CD4 T cell-depleted PBMC and LNMC were suspended in R-10 medium and used the same day in ELISPOT assays. Isolated lymph node CD4 T cells were pelleted and used for RNA extraction and cell-associated viral RNA quantitation and sequencing.

Adnan S., Colantonio A.D., Yu Y., Gillis J., Wong F.E., Becker E.A., Piatak M J.r., Reeves R.K., Lifson J.D., O’Connor S.L, & Johnson R.P. (2015). CD8 T Cell Response Maturation Defined by Anentropic Specificity and Repertoire Depth Correlates with SIVΔnef-induced Protection. PLoS Pathogens, 11(2), e1004633.

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Activation and Expansion of T Cells

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CD3 antibody (eBioscience, cat.no. 16-0037-85) was diluted 1:200 in sterile PBS and used to coat T75 flasks or wells in 12- or 24-well plates for 1 h at 37 °C. The antibody solution was then removed and the flasks or wells were washed three times with sterile 1× PBS. PBMCs were diluted in the growth medium to 10 to 20 × 10⁶ cells/flask with 20 mL of the final volume or at a cell density of 2 × 10⁵ to 1 × 10⁶ cells/mL in 12- or 24-well plates. CD28 antibody (eBioscience, cat.no. 16-0289-85) at a concentration of 1 µg/mL was added to the flask or wells. The flasks or wells were further incubated for 3 days before running experiments. This protocol was also used in the experiments with pure CD4⁺ T cells (>99%) isolated from PBMC with CD4 Dynabeads® in combination with Detachabead^®reagent (Dynal, Oslo, Norway). For comparison, cells were also activated overnight with PHA (5 µg/mL) or cell stimulation cocktail (CSC, 1×), a mixture of phorbol 12-myristate 13-acetate (PMA) and ionomycin (eBioscience, cat.no. 00-4970-93). In some experiments, expansion of a T-cell population was stimulated by using recombinant IL-2 [33 (link)]. After coating flasks/wells with anti-CD3 antibody, recombinant IL-2-human (10 U/mL) (Sigma-Aldrich, cat.no. 57600-1VL) was used to activate the T cells. The flasks or wells were further incubated for 3 days before experiments.

Darvekar S., Juzenas P., Oksvold M., Kleinauskas A., Holien T., Christensen E., Stokke T., Sioud M, & Peng Q. (2020). Selective Killing of Activated T Cells by 5-Aminolevulinic Acid Mediated Photodynamic Effect: Potential Improvement of Extracorporeal Photopheresis. Cancers, 12(2), 377.

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Isolation and Enrichment of Mouse T Cells

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T cells from donor mice were isolated from LNs and spleen by passing tissue through a 40-μm filter to obtain single cell suspension. This cell suspension was treated with ACK lysis buffer to deplete red blood cells (RBCs). OT-I x CCR5^+/+ mice are on a Rag2^−/− background, and T cells isolated from these mice were 75–80% CD8⁺ without additional enrichment steps. CD8⁺ T cells from OT-I x CCR5^−/− and C57BL/6 mice were enriched using negative selection with B220 and CD4 Dynabeads (Life Technologies, Grand Island, NY). OT-II T cells were enriched using negative selection with B220 and CD8 Dynabeads.

Askew D., Su C.A., Barkauskas D.S., Dorand R.D., Myers J., Liou R., Nthale J, & Huang A.Y. (2016). Transient surface CCR5 expression by naïve CD8+ T cells within inflamed lymph nodes is dependent on HEV interaction and augments helper T-cell dependent memory response. Journal of immunology (Baltimore, Md. : 1950), 196(9), 3653-3664.

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CFSE-based T cell Proliferation Assay

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For CFSE dilution assays, splenic and LN CD4⁺ OT‐II T cells were purified using CD4⁺ Dynabeads (Life Technologies) following the manufacturer's protocol. T cells were labeled with 5 μM CFSE (Life Technologies) for 15 min at 37°C, prior to culture with 5 × 10⁴ WT or Ifnar1^−/− FLDCs in the presence of 0.01 μg/ml OVA^323–339 peptide (CRB) or 5 μg/ml OVA protein (Sigma) which had been endotoxin depleted in‐house. Cultures were incubated at 37°C for 4 days prior to assessment of CFSE dilution by flow cytometry. In vitro co‐culture polarization experiments were performed with 5 × 10⁴ KN2xIL‐13eGFP⁻CD4⁺ or KN2xIL‐10eGFP⁻CD4⁺ T cells which were cultured in 96‐well plates for 4 days with 2,500 WT or Ifnar1^−/− FLDCs, 1 μg/ml soluble anti‐CD3, and with or without rIL‐4 (20 ng/ml; PeproTech; Cook et al, 2015). T cell expression of huCD2 (IL‐4; Life Technologies) or eGFP (IL‐10 or IL‐13) was then assessed by flow cytometry.

Webb L.M., Lundie R.J., Borger J.G., Brown S.L., Connor L.M., Cartwright A.N., Dougall A.M., Wilbers R.H., Cook P.C., Jackson‐Jones L.H., Phythian‐Adams A.T., Johansson C., Davis D.M., Dewals B.G., Ronchese F, & MacDonald A.S. (2017). Type I interferon is required for T helper (Th) 2 induction by dendritic cells. The EMBO Journal, 36(16), 2404-2418.

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Isolation and Transfer of Regulatory T Cells

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CD4⁺CD25⁺ T reg cells (>90% pure) were isolated from spleens and LNs of CD45.1⁺ WT, CD45.2⁺Ifnar1^−/−, or Ifnar1^−/−Cd28^−/− mice by magnetic separation using CD4 Dynabeads (Invitrogen) and CD25 microbeads (Miltenyi Biotech). 5–10 × 10⁵ CD4⁺CD25⁺ T reg cells were injected intravenously into each recipient CD45.1⁺CD45.2⁺ mouse. For transfer of CD44^hi T reg cells, Foxp3^GFP mice were pretreated with IL-2 complex before T reg cell isolation: 50 µg anti–IL-2 (JES6; BioXCell) was incubated with 1.5 µg recombinant murine IL-2 (carrier free; eBioscience) in PBS overnight at 4°C and injected into donor mice intraperitoneally on days 0, 2, and 4. Mice were sacrificed on day 6 for sorting of CD4⁺Foxp3^GFPCD44^hiCD62L^lo T reg cells. 5 × 10⁵ sorted T reg cells were transferred per mouse. Mice were infected 1 d after transfer. For IL-2, ICOSL, and B7-1/B7-2 blockade, recipient mice were injected intraperitoneally with a mixture of 100 µg anti–IL-2 (JES6; BioXCell) and 100 µg anti-IL-2 (S4B61; BioXCell), with 100 µg anti-ICOSL (HK5.3; BioXCell), with a mixture of 100 µg anti–B7-1 (16-10A1; BioXCell) and 100 µg anti–B7-1 (GL-1; BioXCell), or with 100 µg rat IgG (Sigma-Aldrich) before T reg cell transfer (day −1) and every 2 d thereafter.

Srivastava S., Koch M.A., Pepper M, & Campbell D.J. (2014). Type I interferons directly inhibit regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection. The Journal of Experimental Medicine, 211(5), 961-974.

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Regulatory T Cell-Mediated Modulation of Islet Inflammation

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The human islets were dissociated into single cells with 0.05% trypsin-EDTA. The single cells (0.5 million cells) were cultured with or without 1 million ex vivo expanded T_regs in the presence of CD3/CD28 beads in 10% HS RPMI-1640 medium for 48 hours. LPS (1 µg/mL) was then added to the cultures for an additional 24 hours. As a control, the islets were co-cultured with autologous T_effs under the same conditions. After the incubation period, T_regs or T_effs were positively isolated using CD4 Dynabeads (Invitrogen). The eluted fraction was used for flow cytometric analysis and real-time RT-PCR for expression of MCP-1.

Xiao F., Ma L., Zhao M., Huang G., Mirenda V., Dorling A., Lechler R, & Lombardi G. (2014). Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice. PLoS ONE, 9(3), e90387.

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Splenic Cell Stimulation Assay

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Splenic cells (2.4 × 10⁵) were stimulated with Pokeweed mitogen (PWM, 40 μg/ml, Sigma, St Louis, MO), human IL-2 (15 U/ml), IL-4 (100 U/ml) (Life Technologies), and human CD3 and CD28 Abs (1 μg/ml each, BD Biosciences). At day 7 of culture, half of the medium was replaced by fresh medium. Immunoglobulin secretion in supernatants at day 16 of culture was measured by an enzyme-linked immunosorbent assay (ELISA) (human IgG ELISA quantitation set; Bethyl Laboratories,). For co-cultures, negatively isolated human CD4 T cells (7.5 × 10⁴) from DRAG mice (CD4 dynabeads, Invitrogen) were added to the splenic cells of A2 mice and stimulated as above.

Majji S., Wijayalath W., Shashikumar S., Pow-Sang L., Villasante E., Brumeanu T.D, & Casares S. (2016). Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice. Scientific Reports, 6, 28093.

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Isolation and Activation of CD4+ T Cells

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CD4⁺ T cells were isolated and stimulated as previously described^{10 (link)}. Briefly, CD4⁺ T cells were isolated from whole blood by negative selection using RosetteSep human CD4⁺ T cell enrichment cocktail (STEMCELL Technologies Inc., Vancouver, BC) and RosetteSep density medium gradient centrifugation. Isolated CD4⁺ T cells were placed in freezing container at −80°C for overnight, and then moved into a liquid nitrogen tank for long-term storage. On the day of activation, CD4⁺ T cells were thawed in a 37°C water bath, counted and resuspended in RPMI-1640 supplemented with 10% FCS, and plated at 50,000 cells per well in a 96 well round-bottom plate. Cells were either left untreated or stimulated with beads conjugated with anti-CD3 and anti-CD28 antibodies (Dynabeads, Invitrogen #11131D, Life Technologies) at a cell:bead ratio of 1:1 for 48 hours, a time point we previously found to maximize the gene expression response in CD4⁺ T cells. At each time point, cells were further purified by a second step positive selection with CD4⁺ Dynabeads (Invitrogen #11145D, Life Technologies).

Gate R.E., Cheng C.S., Aiden A.P., Siba A., Tabaka M., Lituiev D., Machol I., Gordon M.G., Subramaniam M., Shamim M., Hougen K.L., Wortman I., Huang S.C., Durand N.C., Feng T., De Jager P.L., Chang H.Y., Aiden E.L., Benoist C., Beer M.A., Ye C.J, & Regev A. (2018). Genetic determinants of co-accessible chromatin regions in activated T cells across humans. Nature genetics, 50(8), 1140-1150.

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CFSE-Based T Cell Proliferation Assay

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Purified CD4⁺ T cells (CD4 T cells beads, Miltenyi or CD4 Dyna Beads, Invitrogen) were labeled with CFSE or CellTrace Volet (CTV) (Thermofisher) according to manufacturer’s protocol and cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (FBS, GIBCO Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (all from GIBCO) together with DC and irradiated feeder cells (40 Gy) in 2:1:1 ratio for 72 or 96h. Polyclonal CD4⁺ T cells were stimulated with αCD3 (2C11) and αCD28 (37.51) (BioXcell); OT-II cells with OVA_323-339 (ISQAVHAAHAEINEAGR) peptide (Pepscan) and Trp1 cells with Trp1_106-130 (SGHNCGTCRPGWRGAACNQKILTVR) peptide (Pepscan) at concentration indicated in the Figure 2. Cells were additionally supplemented with IL-2, IL-15 or IL-7 (Peprotech) at a concentration indicated in the Figure 2. Mouse CD4⁺ T cells were cultured with αCD25 (PC61, BioXcell) and αIL-2 (JES6-1A12, BioXcell), added to the culture 24h post stimulation with αCD3 and αCD28 in a concentration of 5 μg/ml.

Śledzińska A., Vila de Mucha M., Bergerhoff K., Hotblack A., Demane D.F., Ghorani E., Akarca A.U., Marzolini M.A., Solomon I., Vargas F.A., Pule M., Ono M., Seddon B., Kassiotis G., Ariyan C.E., Korn T., Marafioti T., Lord G.M., Stauss H., Jenner R.G., Peggs K.S, & Quezada S.A. (2020). Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4+ T Cells. Immunity, 52(1), 151-166.e6.

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Tracking T cell responses in vivo

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CD4⁺ T cells from CD45.1⁺2⁺ TSLPR-WT or CD45.2⁺ TSLPR-KO donors, both expressing the 4C13R double reporter, were enriched by positive selection, using either CD4 Dynabeads (Invitrogen) or CD4 microbeads with an autoMACS Pro Separator (Miltenyi Biotec), and injected i.v. into OTII mice back-crossed onto a CD45.1⁺ background. OTII-recipient mice were used as their response to MC903 is reduced compared with C57BL/6 mice, thus allowing a more sensitive detection of transferred T cells. Similar results but lower donor cell recoveries were obtained using B6-SJ recipients. MC903 treatment was started 1 d after T cell transfer, and mice were killed on day 8.

Ochiai S., Jagot F., Kyle R.L., Hyde E., White R.F., Prout M., Schmidt A.J., Yamane H., Lamiable O., Le Gros G, & Ronchese F. (2018). Thymic stromal lymphopoietin drives the development of IL-13+ Th2 cells. Proceedings of the National Academy of Sciences of the United States of America, 115(5), 1033-1038.

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