Anti human cd28

Manufactured by BioLegend

Sourced in United States

Anti-human CD28 is a monoclonal antibody that binds to the CD28 receptor on the surface of T cells. CD28 is a co-stimulatory molecule that plays a critical role in the activation and proliferation of T cells. This antibody can be used in various applications to study T cell function and immune responses.

Automatically generated - may contain errors

Lab products found in correlation

Anti human cd3, by BioLegend (12 mentions) Magnetic microbeads, by BD (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Cck 8, by Beyotime (1 mentions) Anti human cd28, by Miltenyi Biotec (1 mentions) Anti il 4, by Miltenyi Biotec (1 mentions) Anti ifnγ, by Miltenyi Biotec (1 mentions) 2 nbdg, by Thermo Fisher Scientific (1 mentions) Anti mouse cd3, by BioXCell (1 mentions) Anti mouse cd28, by BioXCell (1 mentions) Cfse proliferation dye, by Thermo Fisher Scientific (1 mentions) Aqua live dead viability dye, by Thermo Fisher Scientific (1 mentions) Easysep human cd14 positive selection kit, by STEMCELL (1 mentions) Easysep t cell enrichment kit, by STEMCELL (1 mentions) Recombinant human macrophage colony stimulating factor m csf, by BioLegend (1 mentions) Recombinant human il 2, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by ProSpec (1 mentions) Clone okt3, by BioLegend (1 mentions) Clone cd28, by BioLegend (1 mentions) Penicillin streptomycin, by American Type Culture Collection (1 mentions)

Close spelling variants of this product

Mouse anti-human CD28 antibody Anti-human CD28 (clone CD28.2) Anti-human CD3/CD28 antibodies Soluble anti-human CD28 Mouse anti-human CD28 Ultra-LEAF purified anti–human CD28 antibody Anti-human CD28 antibody Anti-human CD28 mAb Anti-CD28

15 protocols using anti human cd28

TCR Engineered T Cell Activation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

The MAGE-A3+HLA-A2+ PC9 and A375 cells were treated with 20 µg/mL mitomycin C for 30 min and transferred to 48/96-well plates. J76 cells expressing the exogenous TCR-1, TCR-2, TCR-12, TCR-103 and TCR-207 TCRs were co-cultured with PC9 cells at a 2:1 ratio (2×10⁵: 1×10⁵, 48-well) or A375 cells at a 2:1 ratio (0.5×10⁵: 0.25×10⁵, 96-well), respectively, and stimulated with 1 µg/mL anti-human CD28 (BioLegend, Cat#302901, US), 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2 (125 Ala) Injection). T cell activation marker CD69 (BioLegend, Cat#310909, US) and CD137 (BioLegend, Cat#309809, US) were detected at 16 h. Hoechst staining was used to detect the living PC9 or A375 cells at 96 h. The ImageJ software was used to count the living cells.

Zhang B., Ren Z., Zhao J., Zhu Y., Huang B., Xiao C., Zhang Y., Deng J., Mao L., Tang L., Lan D., Gao L., Zhang H., Chen G, & Luo O.J. (2023). Global analysis of HLA-A2 restricted MAGE-A3 tumor antigen epitopes and corresponding TCRs in non-small cell lung cancer. Theranostics, 13(13), 4449-4468.

+ Open protocol

+ Expand

Isolation and Activation of T Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human CD3⁺, CD4⁺, and CD8⁺ T cells were isolated with magnetic microbeads (BD, 552593, 557766, and 557767), respectively. The purity of the enriched subset was validated by flow cytometry and was generally higher than 95% (CD3⁺, CD4⁺, and CD8⁺/CD3 T cells). Cells were activated with plate bound anti-human CD3 (5 µg/mL; BioLegend, 300314) and anti-human CD28 (1 µg/mL; BioLegend, 302923) for 48 hours in the presence of 400 U/mL IL-2. The medium was refreshed on day 2. CD3⁺, CD4⁺, and CD8⁺ T cells were cultured in RPMI-1640 medium and supplemented with 100 U/mL IL-2, and 10% FBS (Gibco, 10 270–106). Unless mentioned otherwise, CD3⁺, CD4⁺, and CD8⁺ T cells used in all the experiments were pretreated with 1α,25(OH)₂D₃ (50 nM) or vehicle two times at 1-day intervals.

Li P., Zhu X., Cao G., Wu R., Li K., Yuan W., Chen B., Sun G., Xia X., Zhang H., Wang X., Yin Z., Lu L, & Gao Y. (2022). 1α,25(OH)2D3 reverses exhaustion and enhances antitumor immunity of human cytotoxic T cells. Journal for Immunotherapy of Cancer, 10(3), e003477.

+ Open protocol

+ Expand

Cytotoxicity assay of TCR-engineered T cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The MAGE-A3+HLA-A2+ PC9 and A375 cells were treated with 20 µg/mL mitomycin C for 30 min and inoculated to 96-well plates (1×10⁴/well, 2.5×10⁴/well, respectively). J76 cells expressing the exogenous TCR-1, TCR-2, TCR-12, TCR-103 and TCR-207 TCRs were co-cultured with PC9 or A375 cells at a 2:1 ratio, respectively, and stimulated with 1 µg/mL anti-human CD28 (BioLegend, Cat#302901, US) and 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2 (125 Ala) Injection). After co-cultivation for 96 h, the OD values were detected by the CCK8 assays (Beyotime, Cat#C0038, China) according to the manufacturer's instructions.

+ Open protocol

+ Expand

Differentiation of T Helper Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols

For differentiation into T helper cell 1/2 subsets, CD4⁺ T cells were incubated for 96 h in cell culture plates covered with anti‐human CD3 (#300437, BioLegend, San Diego, CA) using TexMACS^TM medium. Cells were stimulated with T Cell TransAct^TM human as described above. Specific cytokines were added for Th1 differentiation (anti‐human CD28 [0.5 µg/ml; #302933, BioLegend, San Diego, CA], IL2 [100 U/ml], IL12 [50 ng/µl], and anti‐IL4 [20 ng/µl; #130 097 742 | #130‐096‐704 | # 130‐095‐709, Miltenyi Biotec, Bergisch Gladbach, Germany]) and for Th2 differentiation [anti‐human CD28 (0.5 µg/ml), IL2 (100 U/ml), IL4 (50 ng/ml), anti‐IFNγ (50 ng/ml), and anti‐IL12 (50 ng/ml; #130 097 742 | #130‐096‐753 | #130 095‐743 | # 130‐103‐738, Miltenyi Biotec, Bergisch Gladbach, Germany)], respectively. After 4 days, the cell suspensions were transferred to an uncovered cell culture plate and incubated for another 2 days with the same supplements except anti‐human CD28.

Hirschberger S., Strauß G., Effinger D., Marstaller X., Ferstl A., Müller M.B., Wu T., Hübner M., Rahmel T., Mascolo H., Exner N., Heß J., Kreth F.W., Unger K, & Kreth S. (2021). Very‐low‐carbohydrate diet enhances human T‐cell immunity through immunometabolic reprogramming. EMBO Molecular Medicine, 13(8), e14323.

+ Open protocol

+ Expand

Cytokine-Mediated T Cell Polarization

Check if the same lab product or an alternative is used in the 5 most similar protocols

Mouse and human cytokines used for in vitro polarizations as well as anti-human CD3 and anti-human CD28 were purchased from Biolegend. Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell. Anti-CK2α and anti-CK2β antibodies for detection by flow cytometry were purchased from AbCam and Calbiochem, respectively. Secondary antibodies for flow cytometry were purchased from Jackson ImmunoResearch. Flow cytometry antibodies against mouse CD4, IL-17A, IFN-γ, CD25 and CD45.1 and human CD3, CD4 and Foxp3 were purchased from Biolegend. Flow cytometry antibodies against mouse CD44, Foxp3 and GM-CSF and human IL-17A were purchased from eBioscience. Aqua Live/Dead Viability dye, CFSE proliferation dye and 2-NBDG were purchased from ThermoFisher Scientific. Flow cytometry antibodies and isotype controls for phosphorylated S6, Akt, STAT3, STAT5 and SMAD2/3 were purchased from Cell Signaling. Immunoblotting antibodies against phosphorylated T389, S371 and total S6 Kinase p70 and phosphorylated S129, S473 and total Akt were purchased from Cell Signaling, and antibody against mouse β-actin was purchased from abcam.

Gibson S.A., Yang W., Yan Z., Liu Y., Rowse A.L., Weinmann A.S., Qin H, & Benveniste E.N. (2017). Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis. Journal of immunology (Baltimore, Md. : 1950), 198(11), 4244-4254.

+ Open protocol

+ Expand

Isolation and Differentiation of Macrophages and T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density centrifugation from fresh whole blood donated by healthy volunteers. Human monocytes were purified from PBMCs by EasySep Human CD14 Positive Selection Kit (StemCell Technologies; Cat No.17858) and human naïve CD3⁺ T cells were purified by EasySep T cell enrichment kits (StemCell Technologies; Cat No. 19751). The purity of monocytes and naïve T cells were > 97%, as confirmed by flow cytometry. CD14⁺ monocytes were maintained in RPMI-1640 supplemented with 10% FBS and 100 ng/mL recombinant human macrophage colony stimulating factor (M-CSF) (Biolegend; Cat No. 574802) for 7 days to generate macrophages. For induction of TAMs, macrophages were seeded in 12-well plates and stimulated with 1ml mixture of CM and FBS-containing medium (1:1) for 24 h. Human naïve T cells were first activated using anti-human CD3 (Biolegend; Cat No.317326) pre-coated 6-well plates and then cultured with RPMI-1640 containing 10% FBS, 1 μg/mL anti-human CD28 (Biolegend; Cat No.302914) and 100 IU/mL recombinant human IL-2 (Pepro Tech; Cat No.2000250).

Chen X., Gao A., Zhang F., Yang Z., Wang S., Fang Y., Li J., Wang J., Shi W., Wang L., Zheng Y, & Sun Y. (2021). ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation. Theranostics, 11(7), 3392-3416.

+ Open protocol

+ Expand

Activation of CD8+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD8⁺ T cells were isolated with magnetic microbeads (BD, 557766) from PBMCs of healthy donors (purity of CD8⁺/CD3⁺ T cells > 98%), then were stimulated with plate bound 5 μg/mL anti-human CD3 (BioLegend, 300314) and 1 μg/mL anti-human CD28 (BioLegend, 302934) in 48 wells for 48 h. CD8⁺ T cells were cultured in fresh RPMI-1640 medium supplemented with 10% FBS, 100 U/mL rhIL-2, and incubated at 37°C in a humidified atmosphere of 5% CO₂.

Zhu X., Li K., Liu G., Wu R., Zhang Y., Wang S., Xu M., Lu L, & Li P. (2023). Microbial metabolite butyrate promotes anti-PD-1 antitumor efficacy by modulating T cell receptor signaling of cytotoxic CD8 T cell. Gut Microbes, 15(2), 2249143.

+ Open protocol

+ Expand

Suppressing Jurkat Cell Activation with AML Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the experiment of HL-60 GFP and cS5 cells suppressing Jurkat cell activation, AML cells were cultured for 24 h, and the CM was collected after centrifugation. Jurkat cells were transferred to 24-well cell culture plate which was previously coated with 2.5 µg/mL anti-human CD3 (Biolegend, 317326) followed by supplying with 0.5 µg/mL anti-human CD28 (Biolegend, 302934) with/without HL-60 GFP and cS5 cells in 1 mL culture medium for 24 h. For the experiment of STAT5 suppressing Jurkat cell or PBMC activation by lactate-facilitated PD-L1 expression, 96-well cell culture plate was coated with 2.5 µg/mL anti-human CD3 overnight. Subsequently, Jurkat cells or PBMCs were mixed with AML cells in cell culture plate and stimulated by 0.5 µg/mL anti-human CD28 in 200 μL culture medium for 24 h. The activation of Jurkat cells and PBMCs were analyzed by flow cytometry.

Huang Z.W., Zhang X.N., Zhang L., Liu L.L., Zhang J.W., Sun Y.X., Xu J.Q., Liu Q, & Long Z.J. (2023). STAT5 promotes PD-L1 expression by facilitating histone lactylation to drive immunosuppression in acute myeloid leukemia. Signal Transduction and Targeted Therapy, 8, 391.

+ Open protocol

+ Expand

Isolation and Activation of Primary T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were isolated from the peripheral blood of healthy donors using Ficoll-Paque density gradient centrifugation. T cells were activated with plate-bound anti-human CD3 (2 µg/mL, clone OKT3) and anti-human CD28 (2 µg/mL, clone CD28.2) antibodies (BioLegend, San Diego, CA, USA) in the presence of 100 U/mL interleukin-2 (PROSPEC, East Brunswick NJ, USA). T cells were cultured in 25 cm² cell culture flasks with iMediam for T medium (GC LYMPHOTEC, Tokyo, Japan) in a total volume of 10 mL and maintained at 37 °C in 5% CO₂ atmosphere. The cells were diluted up to 5 × 10⁵ T cells/mL by adding fresh culture medium supplemented with 25 U/mL interleukin-2 on days 4, 7, and 10. On day 14, the T cells were used for co-culture with T3M-1 Clone2 cells. All protocols and experiments involving primary PBMCs were approved by the Institutional Review Board of the Showa University. Informed consent was obtained from all volunteers.

Murayama M., Hosonuma M., Kuramasu A., Kobayashi S., Sasaki A., Baba Y., Narikawa Y., Toyoda H., Isobe J., Funayama E., Tajima K., Sasaki A., Maruyama Y., Yamazaki Y., Shida M., Hamada K., Hirasawa Y., Tsurui T., Ariizumi H., Ishiguro T., Suzuki R., Ohkuma R., Kubota Y., Horiike A., Sambe T., Tsuji M., Wada S., Kobayashi S., Shimane T., Tsunoda T., Kobayashi H., Kiuchi Y, & Yoshimura K. (2024). Isobutyric acid enhances the anti-tumour effect of anti-PD-1 antibody. Scientific Reports, 14, 11325.

+ Open protocol

+ Expand

Culture and Activation of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

THP-1 cells (ATCC Cat# TIB-202) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin–streptomycin (Gibco), and 50 µM 2-mercaptoethanol. Hepa1-6 (ATCC Cat# CRL-1830) and HEK293T (ATCC Cat# CRL-1573) cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. Isolated PBMCs were cultured in 45% RPMI 1640 medium supplemented with 45% X-VIVO 15 (Lonza),10% FBS, and 1% penicillin–streptomycin. T cells were stimulated with anti-human-CD3 (Biolegend Cat# 317,302 RRID: AB_571927) plus anti-human-CD28 (Biolegend Cat#302,902 RRID: AB_314304) antibodies, and 100 IU/ml recombinant human interleukin-2 was added. The cells were checked routinely for mycoplasma contamination.

Tu X., Chen L., Zheng Y., Mu C., Zhang Z., Wang F., Ren Y., Duan Y., Zhang H., Tong Z., Liu L., Sun X., Zhao P., Wang L., Feng X., Fang W, & Liu X. (2024). S100A9+CD14+ monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function. Journal of Experimental & Clinical Cancer Research : CR, 43, 72.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!