Anti cd3 and anti cd28

Manufactured by BD

Sourced in United States, Germany

The Anti-CD3 and Anti-CD28 products are laboratory reagents used in cell biology research. They are designed to activate and stimulate T cells in vitro. The Anti-CD3 component binds to the CD3 complex on the surface of T cells, while the Anti-CD28 component binds to the CD28 receptor, providing the necessary signals for T cell activation and proliferation. These reagents are commonly used in the study of T cell function, immune response, and related cellular processes.

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12 protocols using anti cd3 and anti cd28

Modulation of T Cell Proliferation by MSC Secretome

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Mouse MSCs (2.5 × 10⁴ cells) were plated to a 24-well plate (Corning) and cultured for 48 h. MSCs-derived supernatant after 72 h culture was prepared as conditioned medium (MSC-CM). Mouse splenocytes were washed twice with phosphate-buffered saline containing 3% FCS and then incubated with an anti-mouse CD3 antibody (BD Pharmingen) at 4°C for 30 minutes. Pure CD3⁺ T cells were sorted by flow cytometry (Influx), and 2.5 × 10⁶ T cells were added to each well at a final concentration of 2.5 × 10⁶ T cells per mL in standard Roswell Park Memorial Institute 1640 medium (1 × RPMI with 10 mg/mL glycine, 100 U/mL penicillin, 100 U/mL streptomycin, and 10% (vol/vol) FBS). For T cells proliferation assays, 5,6-carboxyfluorescein diacetatesuccinimidyl ester (CFSE; Invitrogen) staining (5μmol/L) was used. To activate T cells, anti-CD3 and anti-CD28 (BD Pharmingen; final concentration, 500 ng/mL) were added to T cells cultures. After 3 days of activation in the presence of MSCs or MSC-CM, the CD3⁺ T cells were collected and analyzed by flow cytometry. To investigate the ability of MSCs to inhibit the CD4⁺ and CD8⁺ T-cell subpopulations, the collected cells were also stained with an anti-CD4 and -CD8 antibody (eBioscience).

Feng Y., Liao Y., Huang W., Lai X., Luo J., Du C., Lin J., Zhang Z., Qiu D., Liu Q., Shen H., Xiang A.P, & Zhang Q. (2018). Mesenchymal stromal cells-derived matrix Gla protein contribute to the alleviation of experimental colitis. Cell Death & Disease, 9(6), 691.

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Cytokine Profiling in Immune Cell Activation

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Recombinant human IFN-γ, IL-17, IL-22, IL-23, TNF-α, IL-6 and IL-8 were purchased from R&D Systems (Minneapolis, MN). Anti-human IFN-γ, anti-IL-17, anti- IL-22, anti-IL-23, anti-IL-6, anti-IL-8 and anti-KIM-1 antibodies were purchased from R&D Systems. Anti-CD3 and anti-CD28 were obtained from BD Biosciences (San Diego, CA), and 1,25(OH)₂D3 was obtained from Sigma (St. Louis, MO).

Chung B.H., Kim B.M., Doh K.C., Cho M.L., Kim K.W, & Yang C.W. (2017). Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4+ T cell induced injury in human renal proximal tubular epithelial cells. PLoS ONE, 12(2), e0172536.

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Multilineage Differentiation Potential of MSCs

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Cell media MCDB201, SMEM, DMEM, and RPMI 1640 were obtained from Life Technologies, human antibodies CD14-FITC, CD29-FITC, CD34-FITC, CD44-FITC, CD45-FITC, and CD90-FITC were from GeneTex, CD73-FITC and HLA-DR-PE were from eBioscience, mouse anti-human IgG1-FITC, IgG2a-FITC, and IgG2a-PE were from GeneTex, anti-CD3 and anti-CD28 were from BD Biosciences, Hoechst 33342, Lysotracker Green DND-265, and 6-carboxyfluorescein N-succinimidyl ester (CFSE) were from Invitrogen, Alizarin Red S, Alcian Blue, Oil Red O, phosphate-buffered saline (PBS), human serum albumin (HSA), erythrocyte lysis buffer, and all other chemicals were from Sigma-Aldrich and used without further purification.

Su L.J., Wu M.S., Hui Y.Y., Chang B.M., Pan L., Hsu P.C., Chen Y.T., Ho H.N., Huang Y.H., Ling T.Y., Hsu H.H, & Chang H.C. (2017). Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs. Scientific Reports, 7, 45607.

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Isolation and Characterization of Tregs from Colon Cancer Patients

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Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of colon cancer patients by density-gradient centrifugation with Ficoll. Then, CD4⁺ T cells were separated from PBMC using Dynabeads™ CD4 Positive Isolation Kit (Thermo Fisher Scientific) according to the manufacturers’ instruction. The purity of the sorted CD4⁺ T was verified by flow cytometry. CD4⁺ T cell suspension was stained with CD4-FITC (Abcam) at 4 °C for 30 min. Then, CD4⁺ T cells were gated with CD4 and SSC, the purity of CD4⁺ T cells was examined using a FACSCalibur (BD Biosciences).
CD4⁺ T cells and Tregs were cultured in RPMI 1640 medium containing 10 % FBS and 1 % penicillin/streptomycin at 37 °C and 5 % CO₂. For activation of CD4⁺ T cells, sorted naive CD4⁺ T cells were activated with anti-CD3 and anti-CD28 (BD Biosciences). For differentiation of Tregs, CD4⁺ T cells were incubated with TGF-β (5.0 ng/mL) for 5 days. Tregs were stained with CD25-FITC and Foxp3-PE antibodies to estimate the Treg differentiation efficiency by flow cytometry. Subsequently, Tregs were transfected with LV-IRF4, LV-ctrl, LV-sh-IRF4 or LV-shRNA. Then, the cell culture medium of these modified Tregs was collected by centrifugation, and then incubated with SW480 or HCT116 cells.

Wang J., Li S., Li H., Zhou X., Wen H, & Lai B. (2021). IRF4 overexpression promotes the transdifferentiation of tregs into macrophage‐like cells to inhibit the development of colon cancer. Cancer Cell International, 21, 58.

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Naive T Cell Differentiation Protocols

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Fluorescence-activated cell sorting (FACS)-sorted naive CD4⁺CD25⁻CD62L^hiCD44^lo T cells from splenocytes and lymph node cells of WT and Batf KO mice were activated with plate-bound anti-CD3 and anti-CD28 (BD Pharmingen, 2 μg ml⁻¹) and polarized under various Th conditions as described previously33 (link). The culture conditions are as follows: Th0 (50 U ml⁻¹ hIL-2, PeproTech); Th1 (10 ng ml⁻¹ IL-12, PeproTech), 50 U ml⁻¹ hIL-2, 10 μg ml⁻¹ anti-IL-4 (Bioexcel); Th2 (10 ng ml⁻¹ IL-4, PeproTech), 50 U ml⁻¹ hIL-2 and 10 μg ml⁻¹, anti-IFN-γ (Bioexcel); Th17 (20 ng ml⁻¹ IL-6, PeproTech), 2 ng ml⁻¹ TGF-β (PeproTech), 10 μg ml⁻¹ anti-IFN-γ and 10 μg ml⁻¹ anti-IL-4); and iTreg (2 ng ml⁻¹ TGF-β, 10 μg ml⁻¹, anti-IFN-γ and 10 μg ml⁻¹ anti-IL-4). Four days after culture, differentiated cells were restimulated with plate-bound anti-CD3 for 4 h and analysed.

Sahoo A., Alekseev A., Tanaka K., Obertas L., Lerman B., Haymaker C., Clise-Dwyer K., McMurray J.S, & Nurieva R. (2015). Batf is important for IL-4 expression in T follicular helper cells. Nature Communications, 6, 7997.

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PD-1 Inhibitor Protocol for T Cell Activation

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DMEM, RPMI-1640 medium, Leibovitz 15 medium, FBS, GlutaMAX™ and 2-mercaptoethanol were purchased from Gibco; Thermo Fisher Scientific, Inc. Penicillin-streptomycin was purchased from Sigma-Aldrich (Merck KGaA). FuGENE^® HD Transfection Reagent was purchased from Promega Corporation and TRIzol^® reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc). Anti-CD3 and anti-CD28 were purchased from BD Biosciences. Allophycocyanin-conjugated anti-human-CD274/PD-L1 antibody were purchased from eBioscience (Thermo Fisher Scientific, Inc.). Rabbit anti-Erk1/2, rabbit anti-phospho-Erk1/2, rabbit anti-AKT, rabbit anti-GAPDH and horseradish peroxidase-conjugated Goat anti-rabbit immunoglobulin G were purchased from Cell Signaling Technology, Inc. Human IFN-γ ELISA kits and human IL-2 ELISA kits were purchased from Cisbio (PerkinElmer, Inc.). A0-L (a PD-1 inhibitor; patent no. WO 2015/034820 A1; molecular weight, 475.58) was synthesized by Dr Wei Lv of East China Normal University. The company name and catalog number for ELISA kits and all antibodies are listed in Table SI.

Zheng Y., Fang Y.C, & Li J. (2019). PD-L1 expression levels on tumor cells affect their immunosuppressive activity. Oncology Letters, 18(5), 5399-5407.

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Lymphoid Cell Isolation and Stimulation

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Splenocytes and cells were isolated from the gut-draining and head-skin-draining lymph nodes of euthanized mice by passing the organs through a 70-μm-mesh-size cell strainer (BD, USA). The cells were cultured as triplicates of 3.5 × 10⁵ to 5 × 10⁵ cells in 200 μl RPMI 1640 medium (PanBiotech, Germany) containing 10% fetal calf serum, 20 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cultures were stimulated with 40 μg/ml worm or tick Ag, 30 μg/ml B. afzelii Ag (all for 72 h), or 1 μg/ml anti-CD3 and anti-CD28 (BD, USA) for 48 h at 37°C in a 5% CO₂ atmosphere. The supernatants were stored at −20°C for cytokine detection.

Maaz D., Rausch S., Richter D., Krücken J., Kühl A.A., Demeler J., Blümke J., Matuschka F.R., von Samson-Himmelstjerna G, & Hartmann S. (2016). Susceptibility to Ticks and Lyme Disease Spirochetes Is Not Affected in Mice Coinfected with Nematodes. Infection and Immunity, 84(5), 1274-1286.

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Co-culture of Cholangiocytes and T Cells

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Isolated lymphocytes from spleen or liver were cultivated in Panserin 401-Medium (PAN Biotech, Germany) supplemented with 1 % Penicillin-Streptomycin (Thermo Fisher Scientific, Germany) in flat bottom 96 well plates. Cells were seeded at 500,000 cells per well and stimulated for 24 h using anti-CD3 and anti-CD28 (each 2 µg/ml, BD Biosciences, Germany).
Primary female mouse derived cholangiocytes were cultivated as described previously^{31 (link)}. Cholangiocytes were grown confluent in 24 well plates and stimulated with fresh medium containing 10 ng/ml IFNgamma and/or IL-17A (PeproTech, USA) for 24 h. For co-culture experiments 500,000 freshly isolated splenic derived CD8⁺ T cells from female OT-1^wt and OT-1^IL17ko donor mice were added for 48 hours to 70 % confluent primary mouse cholangiocytes isolated from female K14-OVAp donors. Supernatants were harvested and analyzed using ELISA for CCL20, IL-6, IL-17A, IFNgamma (all R&D Systems, USA) and granzyme B (Thermo Fisher Scientific, Germany) and cells were harvested for RNA isolation and qPCR analysis.

Stein S., Henze L., Poch T., Carambia A., Krech T., Preti M., Schuran F.A., Reich M., Keitel V., Fiorotto R., Strazzabosco M., Fischer L., Li J., Müller L.M., Wagner J., Gagliani N., Herkel J., SCHWINGE D, & SCHRAMM C. (2020). IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis via PD-1/PD-L1 interaction. Journal of hepatology, 74(4), 919-930.

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Cytokine Production Profiling of T Cells in Colitis

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Colonic tissue or tumors from AOM/DSS-treated mice were homogenized, and total RNA isolated using the Nucleospin RNA kit (Macherey-Nagel). cDNA synthesis was performed using iScript (BioRad), and the cDNA was then used for quantitative PCR using either SYBR Green Master Mix (Applied Biosystems) or Taqman Gene Expression Assays on the ABI 7900HT. Primers sequences are available upon request.
For measurement of T cell-derived cytokines, total lamina propria cells from day 8 AOM/DSS-treated mice were sorted for CD3⁺NK1.1^- T cells, which excludes NKT and iNKT cells, and then ex vivo stimulated with plate-bound anti-CD3 antibody (10 μg/mL) (BD Biosciences, 500-A2) or anti-CD3 and anti-CD28 (2 μg/mL) (BD Biosciences, clone 37.51), respectively. Stimulation was performed in a 96- well plate using a concentration of 2 million/mL T cells. Supernatants were collected 48 hours later and cytokines were measured by ELISA.
For experiments involving stimulation with the Nod1 ligand KF1B, provided as a gift by Dr. Naohiro Inohara, T cells were isolated by magnetic bead purification of total splenocytes and then stimulated with platebound anti-CD3 (1 μg/mL) with or without KF1B (10 μg/mL). Supernatant was collected 24 hours after stimulation and IFNγ levels measured by ELISA.

Zhan Y., Seregin S.S., Chen J, & Chen G.Y. (2016). Nod1 limits colitis-associated tumorigenesis by regulating IFNγ production. Journal of immunology (Baltimore, Md. : 1950), 196(12), 5121-5129.

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Spleen-Derived T Cell Characterization

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Totalsplenocyteswere obtained from aseptically collected mousespleen described previously74 (link). The cells were harvested in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 5% fetal bovine serum. Mononuclear cells (1 × 10⁶) were cultured without treatment as a negative control or with anti-CD3 and anti-CD28 (BD Pharmingen) for 3 days. The cells were stained with various combinations of fluorescence-tagged antibodies against CD4, CD25, IFN-γ (BD Biosciences), Foxp3, and IL-17 (eBioscience). Before intracellular staining, cells were restimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiSTOP (BD Biosciences). Intracellular staining was performed using a kit (eBioscience) following the manufacturer’s protocol. Stained cells were analyzed on a FACSCalibur apparatus (BD Biosciences).

Park M.J., Lee S.H., Moon S.J., Lee J.A., Lee E.J., Kim E.K., Park J.S., Lee J., Min J.K., Kim S.J., Park S.H, & Cho M.L. (2016). Overexpression of soluble RAGE in mesenchymal stem cells enhances their immunoregulatory potential for cellular therapy in autoimmune arthritis. Scientific Reports, 6, 35933.

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