Anti human cd28 clone cd28.2

Manufactured by BioLegend

Anti-human CD28 (clone CD28.2) is a lab equipment product that detects the CD28 protein, which is expressed on the surface of T cells. The CD28 protein plays a crucial role in the activation and regulation of T cell responses.

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Lab products found in correlation

Anti human cd3 clone okt3, by BioLegend (2 mentions) Anti human cd3 clone hit3a, by BioLegend (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Hek293t, by American Type Culture Collection (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Easysep human cd8 t cell isolation kit, by STEMCELL (1 mentions) Recombinant human il 2 and il 15, by BioLegend (1 mentions) Anti human cd49d clone 9f10, by BioLegend (1 mentions) Easysep human cd4 t cell isolation kit, by STEMCELL (1 mentions) Polyethylenimine (pei), by Polysciences (1 mentions) Ficoll gradient, by GE Healthcare (1 mentions) Protein a sepharose column, by Abcam (1 mentions) Monocyte purification kit, by Miltenyi Biotec (1 mentions) Sypro orange, by Merck Group (1 mentions) Gm csf and il 4, by R&D Systems (1 mentions) Hrp conjugated goat anti human igg h l, by Jackson ImmunoResearch (1 mentions) Freestyle 293 medium, by Thermo Fisher Scientific (1 mentions) Human pbmcs, by AllCells (1 mentions) Dynabeads cd4 positive isolation kit, by Thermo Fisher Scientific (1 mentions) Tmb substrate, by Thermo Fisher Scientific (1 mentions)

5 protocols using anti human cd28 clone cd28.2

Isolation and Activation of CD8+ T Cells

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PBMCs were isolated from EDTA-anticoagulated blood using the lymphocyte separation medium (LTS1077, TBD, China) by Ficoll–Paque density gradient centrifugation. The viability of PBMCs in single-cell suspensions were assessed by using Trypan Blue Staining. Each sample with 1 × 10⁷ viable cells was obtained for scRNA-seq or WES.
CD8⁺ T cells are purified using EasySep™ human CD8⁺ T cell isolation kit (Cat #17953, STEMCELL) according to the manufacturer's instructions. The purified CD8 T cells were stimulated with 5 μg/ml anti-human CD3 (clone OKT3, BioLegend Cat# 317326, RRID: AB_11150592) and 2 μg/ml anti-human CD28 (clone CD28.2, BioLegend Cat# 302934, RRID: AB_11148949) for 3 days, then CD8⁺ T cells were collected for analysis or electroporation.

Xiong H., Cui M., Kong N., Jing J., Xu Y., Liu X., Yang F., Xu Z., Yan Y., Zhao D., Zou Z., Xia M., Cen J., Tan G., Huai C., Fu Q., Guo Q, & Chen K. (2023). Cytotoxic CD161−CD8+ TEMRA cells contribute to the pathogenesis of systemic lupus erythematosus. eBioMedicine, 90, 104507.

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Preparation and Characterization of Cell Lines for HIV Research

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HEK 293T (ATCC), TZM-bl [24 (link), 25 (link)] and Phoenix-Ampho [26 (link)] cell lines were grown in Dubelcco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% (^v/_v) fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 μg/ml) (referred to as DF10 medium). TZM-bl expressing NB-mCh or mCh cell lines were established by transduction of NB-mCh or mCh virus-like particles (VLPs) and then selected by fluorescent activated cell sorter (FACS) for the top 10% of mCherry positive cells by mean fluorescent intensity (MFI).
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor’s buffy coat supplied by Australian Red Cross Blood service using Ficoll density gradient centrifugation. CD4⁺ cells were isolated from the PBMCs by using a magnetic-activated cell sorting human CD4⁺ cell isolation kit (Miltenyi Biotec) as per the manufacturer’s instruction. The selected cells were grown in 6 cm tissue culture dishes and stimulated using plates pre-coated with purified anti-human CD3 (clone HIT3a) and anti-human CD28 (clone CD28.2) antibodies (BioLegend) in RPMI medium supplemented with 20% (^v/_v) FBS and 5 ng/ml interleukin-2 (IL-2) (hereafter called RF20 IL-2) for 2 days. All cells were grown at 37 °C in humidified incubators with 5% CO₂.

Rustanti L., Jin H., Lor M., Lin M.H., Rawle D.J, & Harrich D. (2017). A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes. Virology Journal, 14, 52.

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Intracellular Cytokine Profiling of Activated PBMCs

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We stimulated PBMCs with IL-15 (50 ng/mL) for 48 h and IL-2 (500 IU/mL), anti-CD3 (1 ng/mL), anti-CD28 (1 ng/mL), and anti-CD49d (1 ng/mL) for 6 h to detect intracellular CCL3, CCL4, and CCL5 (31 (link)), and with IL-12 (20 ng/mL), IL-15 (10 ng/mL), and IL-18 (20 ng/mL) for 48 h to detect intracellular IL-2, IFNγ and TNFα (26 (link)). GolgiPlug was added to the medium 6 h before harvest to detect intracellular cytokines. Recombinant human IL-12 and IL-18 were purchased from PeproTech (Rocky Hill, NJ). Recombinant human IL-2 and IL-15, anti-human CD3 (clone OKT3), anti-human CD28 (clone CD28.2), and anti-human CD49d (clone 9F10) antibodies were purchased from BioLegend.

Hu W., Li Y.J., Zhen C., Wang Y.Y., Huang H.H., Zou J., Zheng Y.Q., Huang G.C., Meng S.R., Jin J.H., Li J., Zhou M.J., Fu Y.L., Zhang P., Li X.Y., Yang T., Wang X.W., Yang X.H., Song J.W., Fan X., Jiao Y.M., Xu R.N., Zhang J.Y., Zhou C.B., Yuan J.H., Huang L., Qin Y.Q., Wu F.Y., Shi M., Wang F.S, & Zhang C. (2022). CCL5-Secreting Virtual Memory CD8+ T Cells Inversely Associate With Viral Reservoir Size in HIV‐1−Infected Individuals on Antiretroviral Therapy. Frontiers in Immunology, 13, 897569.

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Isolation and Culture of Primary Human T Cells

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HEK293T (ATCC) and Phoenix-Ampho (38 (link)) cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA) supplemented with 10% (vol/vol) fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (referred to as DF10 medium).
Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor’s buffy coat supplied by the Australian Red Cross Blood Service using Ficoll density gradient centrifugation as previously described (21 (link), 22 (link)). Briefly, CD4⁺ cells were isolated from the PBMCs by using an EasySep human CD4⁺ T cell isolation kit (Stemcell Technologies Australia Pty. Ltd., Tullamarine, VIC, Australia) according to the manufacturer’s instructions. The selected cells were stimulated in a precoated 3-cm tissue culture dish with purified anti-human CD3 (clone HIT3a) and anti-human CD28 (clone CD28.2) antibodies (BioLegend, San Diego, CA) in RPMI medium supplemented with 20% (vol/vol) FBS and 5 ng/ml interleukin-2 (IL-2) (referred to as RF20 IL-2 medium) for 2 days. All cells were maintained at 37°C in humidified incubators with 5% CO₂.

Jin H., Sun Y., Li D., Lin M.H., Lor M., Rustanti L, & Harrich D. (2019). Strong In Vivo Inhibition of HIV-1 Replication by Nullbasic, a Tat Mutant. mBio, 10(4), e01769-19.

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Comprehensive Cell Line Protocol

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NCI-N87, HCC1954, and SKBR3 cells were obtained from the Shanghai Cell Line Bank. RPMI1640 medium (61870036), Freestyle 293 medium, Dynabeads CD4 Positive Isolation Kit (11331D), TMB substrate, and fetal calf serum (FCS) were obtained from Thermo Fisher Scientific. PE-labeled goat anti-human IgG (409304), streptavidin-PE (405203), anti-human-CD3 (clone HIT3a), and anti-human-CD28 (clone CD28.2) were obtained from Biolegend. Trastuzumab and humanized anti-PD-L1 scFV were prepared in house. Cell Counting Kit-8 (CCK8) was obtained from Dojindo Laboratories. Human antibody germline sequence and primers were synthesized by Sangon Biotech. IL-4 and GM-CSF were obtained from R&D Systems, and all other recombinant proteins were products of Sino Biological. SYPRO Orange was obtained from Sigma-Aldrich. Human IFN-γ ELISA Kit (EHC102g) was obtained from Neobioscience. Cytotoxicity detection kit (LDH) was from Roche Life Science. Matrigel was obtained from BD Biosciences. Polyethylenimine (PEI) was obtained from Polysciences. Protein A probes were obtained from Pall Corporation. Protein A-Sepharose Column was obtained from BioVision. HRP-conjugated goat anti-human IgG (H + L) was obtained from Jackson ImmunoResearch. Monocyte purification kit was obtained from Miltenyi Biotec. Ficoll gradient was obtained from GE healthcare. Human PBMCs were obtained from Allcells.

Chen Y.L., Cui Y., Liu X., Liu G., Dong X., Tang L., Hung Y., Wang C, & Feng M.Q. (2021). A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy. The Journal of Biological Chemistry, 297(6), 101420.

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