Anti cd28 clone cd28

Manufactured by BD

Sourced in United States

Anti-CD28 (clone CD28.2) is a monoclonal antibody that recognizes the CD28 cell surface receptor. CD28 is a co-stimulatory molecule expressed on T cells that plays a crucial role in T cell activation and proliferation. The Anti-CD28 (clone CD28.2) antibody can be used as a research tool to study T cell biology and immune system function.

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Lab products found in correlation

Anti cd3 clone ucht1, by BD (5 mentions) T cell purification kit, by Thermo Fisher Scientific (4 mentions) Anti cd3 clone okt3, by Thermo Fisher Scientific (4 mentions) Anti cd3 clone hit3α, by BD (3 mentions) V bottom 96 well plate, by Corning (2 mentions) Anti cd3 clone ucht1, by R&D Systems (2 mentions) Normal medium without glucose, by Thermo Fisher Scientific (2 mentions) Anti cd3 clone hit3a, by BD (2 mentions) Anti cd28 clone 37, by BD (2 mentions) Easysep human cd3 and cd8 t cell isolation kit, by STEMCELL (2 mentions) Easysep mouse cd3 and cd8 t cell isolation kit, by STEMCELL (2 mentions) Ova257 264 peptide, by Merck Group (2 mentions) Anti cd3 clone 145 2c11, by BD (2 mentions) Ls174t, by American Type Culture Collection (2 mentions) Interleukin 2 (il 2), by R&D Systems (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Cd3 and cd8 microbeads, by Miltenyi Biotec (2 mentions) Anti cd8, by BD (1 mentions) Anti cd8 clone bw135 80, by Miltenyi Biotec (1 mentions) Anti cd45ro clone uchl1, by BD (1 mentions)

Close spelling variants of this product

Anti-CD28 (379 citations) Anti-CD28 (clone 37.51, (33 citations) Clone CD28.2 (23 citations) Anti-CD28 mAb (clone L293, (7 citations) Soluble anti-CD28 (clone 37.51, (6 citations) Soluble anti-CD28 (clone CD28.2, (5 citations) Anti-mouse CD28 (clone 37.51) (5 citations) Anti-human CD28 (clone CD28.2, (4 citations) Anti-CD28 antibody (clone 37.51; (3 citations) Hamster anti-mouse CD28 (clone 37.51; (3 citations)

30 protocols using anti cd28 clone cd28

Phenotypic Characterization of Melan-A-Specific T Cells

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Phenotypic characterization on resting T cell clones was performed on 10⁵ T cells labeled with MELOE-1 and Melan-A tetramers (10 μg/mL) (Recombinant protein facility, SFR Santé, Nantes, France), anti-CD8 (clone BW135/80, Miltenyi Biotec), anti-CD45RO (clone UCHL1, BD Biosciences), anti-CD27 (clone M-T271, BD Biosciences), anti-CD28 (clone CD28.2, BD Biosciences), anti-CD62L (clone DREG-56, BD Biosciences), anti-PD-1 (clone EH12, BD Biosciences), anti-CTLA-4 (clone BNI3, Miltenyi Biotec), anti-BTLA (clone J168–540, BD Biosciences), anti-Tim-3 (clone F38–2E2, eBiosciences) and anti-CD95 (clone DX2, BD Biosciences) specific antibodies. PD-1 expression (Clone EH12, BD Biosciences) was tested on specific T cell clones or sorted T cells at rest and after activation by quadruple labeling with specific tetramers, anti-CD8 and anti-CD25 (clone M-A251, BD Biosciences), as activation marker. All the antibodies were used at a concentration of 5 μg/mL. Vß diversity of sorted Melan-A-specific T cell lines was analyzed by labeling with 24 anti-Vß mAbs included in the IOTest Beta Mark TCR V Kit (Beckman-Coulter, IM3497). All the cytometric analyses were performed on a Facs Canto II (BD Biosciences).

Simon S., Vignard V., Florenceau L., Dreno B., Khammari A., Lang F, & Labarriere N. (2015). PD-1 expression conditions T cell avidity within an antigen-specific repertoire. Oncoimmunology, 5(1), e1104448.

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Single-cell analysis of SARS-CoV-2 vaccine-elicited T cells

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For single-cell RNA sequencing, PBMC of three BNT-BNT-BNT-vaccinated, three AZ-BNT-BNT vaccinated, and three BNT-BNT-infected donors were stimulated with 1 µg/ml S-I peptide pool in the presence of purified anti-CD28 (clone CD28.2, BD Biosciences) and anti-CD40 (clone HB14, Miltenyi Biotec). CD4⁺ T cells were enriched by MACS (Miltenyi Biotec) and CD40L⁺4-1BB⁺ CD4⁺ T cells FACS sorted using an FACS Melody (BD). The cells were loaded with a maximum concentration of 1000 cells/µl and a maximum cell number of 17.000 cells on a Chromium Chip G (10x Genomics). Gene expression and TCR libraries were generated according to the manufacturer’s instruction using the Chromium Next GEM single cell 5’Library and Gel bead Kit V1.1 and Chromium Single Cell V(D)J Enrichment Kit for human T cells (10x Genomics). Sequencing was conducted with a NovaSeq 6000 cartridge (Illumina) with 20.000 reads per cell for GEX libraries and 5.000 reads per cell for TCR libraries.

Henze L., Braun J., Meyer-Arndt L., Jürchott K., Schlotz M., Michel J., Grossegesse M., Mangold M., Dingeldey M., Kruse B., Holenya P., Mages N., Reimer U., Eckey M., Schnatbaum K., Wenschuh H., Timmermann B., Klein F., Nitsche A., Giesecke-Thiel C., Loyal L, & Thiel A. (2023). Primary ChAdOx1 vaccination does not reactivate pre-existing, cross-reactive immunity. Frontiers in Immunology, 14, 1056525.

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In vitro Expansion of Virus-Specific CD8+ T Cells

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Around 1–2 × 10⁶ PBMCs were stimulated with epitope-specific peptides (10 μg ml⁻¹) and anti-CD28 (clone CD28.2, 0.5 μg ml⁻¹, BD Biosciences, Germany) in 1 ml complete medium and incubated at 37 °C for 14 days. At day 3, 7 and 10, culture was supplemented with 0.5 ml of fresh medium including 20 IU ml⁻¹ rIL-2 (Miltenyi Biotec, Germany). Tetramer and intracellular cytokine staining were performed at day 14. The expansion index was calculated as follows: [A] The absolute number of virus-specific CD8⁺ T cells added at day 0 of in vitro expansion was calculated based on peptide/MHCI tetramer enrichments (see above). [B] At day 14 of in vitro expansion, the absolute number of expanded virus-specific CD8⁺ T cells was determined based on direct FACS analyses. The expansion index was then calculated as (([B]/[A])+1), allowing subsequent logarithmic calculation despite zero values, resulting in the expansion factor=log(expansion index).

Wieland D., Kemming J., Schuch A., Emmerich F., Knolle P., Neumann-Haefelin C., Held W., Zehn D., Hofmann M, & Thimme R. (2017). TCF1+ hepatitis C virus-specific CD8+ T cells are maintained after cessation of chronic antigen stimulation. Nature Communications, 8, 15050.

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Proliferation of Regulatory T Cells

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To assess proliferation of Tcons, Tcons (CD4⁺CD127^hiCD25^lo) were labeled in 1 μM CFSE solution as previously described (see Ref. 18 (link)). Labeled Tcons were cultured at a constant number of 5 × 10⁴/well either alone (1:0) or at a 1:2, 1:4, and, where cell numbers permitted, 1:8 ratio with either P2-enriched Tregs or P3-enriched Tregs on V-bottom 96-well plates (Costar) precoated with 1 μg/ml anti-CD3 (clone UCHT1, R&D Systems) and 5 μg/ml anti-CD28 (clone CD28.2, BD Biosciences) Abs in culture medium (RPMI 1640 supplemented with 10% FBS and 100 U/ml penicillin/streptomycin) at 37°C and 5% CO₂ for 4–5 d. Final cell concentration was maintained at 1 × 10⁶/ml. A cytokine multiplex assay was performed as previously described (19 (link)).

Bending D., Pesenacker A.M., Ursu S., Wu Q., Lom H., Thirugnanabalan B, & Wedderburn L.R. (2014). Hypomethylation at the Regulatory T Cell–Specific Demethylated Region in CD25hi T Cells Is Decoupled from FOXP3 Expression at the Inflamed Site in Childhood Arthritis. The Journal of Immunology Author Choice, 193(6), 2699-2708.

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Antibody Binding Characterization of TIM3

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Example 7

Total human T cells were isolated from PBMC using a Miltenyi T cell purification kit and activated with plate-bound anti-CD3 (1 μg/ml; Anti-CD3 clone OKT3, eBioscience, Catalog #16-0037-85) and soluble anti-CD28 (1 μg/ml; Anti-CD28 clone CD28.2, BD Biosciences, Catalog #555725) for 4 days. TIM3 was expressed in >80% of T cells, as determined by FACS. The T cells were incubated with various anti-TIM3 antibodies for 30 minutes, followed by incubation with selected biotin-labeled anti-TIM3 antibodies for 30 minutes and detected by PE-conjugated streptavidin. The results, which are shown in FIG. 19, indicate that antibodies 13A3, 3G4, 17C3, 17C8, and 9F6 are in the same binning group (Group I), i.e., cross-compete each other, while antibodies 8B9 and 8C4 are in a separate binning group (Group II), i.e., do not cross-compete with the antibodies in Group I, but cross-compete with each other. The antibodies in binning group I were shown to have biological activity (see Examples), while those in binning group II had weaker activity. Two anti-TIM3 antibodies which did not cross-compete with either Group I or Group II, did not appear to have any biological activity. The antibodies of binning group I were also those that interfered with TIM3 binding to PS (as further described herein).

US10077306B2. Antibodies against TIM3 and uses thereof (2018-09-18). Bristol-Myers Squibb Company [US]. Inventors: Xiao Min Schebye [US], Mark J. Selby [US], Michelle Minhua Han [US], Christine Bee [US], Andy X. Deng [US], Anan Chuntharapai [US], Brigitte Devaux [US], Huiming Li [US], Paul O. Sheppard [US], Alan J. Korman [US], Daniel F. Ardourel [US], Ekaterina Deyanova [US], Richard Huang [US], Guodong Chen [US], Michelle Kuhne [US], Hong-An Troung [US].

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Regulation of T-cell Proliferation by Tregs

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To assess proliferation of Tcon, T con cells (CD4⁺CD127^hiCD25^lo) were labeled in 1μM CFSE solution as previously described, see Pesenacker et al.(18 (link)), Labeled Tcon cells were cultured at a constant number of 5×10⁴/well either alone (1:0) or at a 1:2, 1:4, and where cell numbers permitted, 1:8 ratio with either P2-enriched Treg or P3-enriched Treg on V-bottom 96-well plates (CoStar) pre-coated with 1μg/mL anti-CD3 (clone UCHT1, R&D systems) and 5μg/mL anti-CD28 (clone CD28.2, BD Biosciences) antibodies in culture medium (RPMI 1640 supplemented with 10% FBS and 100U/mL penicillin/streptomycin), at 37°C and 5% CO₂ for 4-5 days. Final cell concentration was maintained at 1×10⁶/ml. Cytokine multiplex assay was performed as previously described (19 (link)).

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Rhesus T Cell Transduction with Retronectin

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Blood was collected from rhesus macaques at TNPRC according to Institutional Animal Care and Use Committee guidelines and the NIH Guide for the Care and Use of Laboratory Animals. Rhesus PBMCs were isolated by discontinuous-density gradient centrifugation using LSM (Lymphocyte Separation Media, MP Biomedicals) and resuspended in RPMI-1640 supplemented with 10% FBS; 50 U/ml penicillin; 50 μg/ml streptomycin; 2 mM L-glutamine; 40 U/ml IL-2. Rhesus T cells (1×10⁶ cells/ml or 0.5 ×10⁶ cells/cm²) were stimulated using plate- bound anti-CD3 (clone 6G12; BD-Pharmingen) and anti-CD28 (clone CD28.2; BD-Pharmingen) for 3 days at 37°C, 5% CO₂.
Wells on a non-tissue culture treated 24-well plates were coated with Retronectin (RN), a recombinant fragment of fibronectin (Takara, Fisher Scientific), at 10 μg per cm² final concentration for 2-3 hrs at room temperature followed by one wash of PBS with 2% BSA for 30 mins. Plates were washed with PBS and were ready to use. RN-coated plates were preloaded with viral supernatants by centrifugation at 2000 rpm for 30 min at 4°C. T cells were seeded on RN-coated plates and exposed to viral particles that remained bound to the RN. Three days post transduction, cells were analyzed for transduction efficiency by flow cytometry.

MacLean A.G., Walker E., Sahu G., Skowron G., Marx P., von Laer D., Junghans R.P, & Braun S.E. (2014). A Novel Real-Time CTL Assay to Measure Designer T Cell Function Against HIV Env+ Cells. Journal of medical primatology, 43(5), 341-348.

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T Cell Phenotyping by Flow Cytometry

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Seven days post co-culture, cells were collected, washed and counted. Single-cell suspensions were then stained with fluorochrome-conjugated antibodies and immunofluorescence was analyzed on a FACS Symphony (BD Biosciences) using standard techniques. Antibodies used in this experiment were: anti-CD3(clone:SK7, BD Biosciences) anti-CD8(clone:SK1, BD Biosciences); anti-CD28 (clone:CD28.2, BD Biosciences); anti-CD62L (clone: DREG56, Biolegend); anti-Ki67 (clone:B56, BD Biosciences); anti-CXCR5(clone:RF8B2, BD Biosciences) and anti-PD-1(Amgen).

Shen S., Sckisel G., Sahoo A., Lalani A., Otter D.D., Pearson J., DeVoss J., Cheng J., Casey S.C., Case R., Yang M., Low R., Daris M., Fan B., Agrawal N.J, & Ali K. (2020). Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity. Frontiers in Immunology, 11, 832.

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Cytokine Profiling of Frozen PBMCs

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Frozen PBMCs were thawed, counted, and resuspended at a density of 15 million live cells per ml in complete RPMI (RPMI with 10% FBS (Gibco) and antibiotics). 100 μl of cell suspension containing 1.5 million cells was added to each well of a 96-well round-bottomed tissue culture plate. The cells were rested overnight at 37 °C in a CO₂ incubator. The next morning, each sample was treated with peptide mega pool (1 μg/ml of each peptide) or single peptide (5 μg/ml) or 0.5% v/v DMSO as negative control in the presence of 1 μg/ml of anti-CD28 (clone CD28.2, BD Biosciences), anti-CD49d (clone 9F10, BD Biosciences), anti-CXCR3 (clone 1C6, BD Biosciences) and anti-CXCR5 (clone RF8B2, BD Biosciences). Peptides were synthesized to 95% purity (Elim Biopharm). All wells contained 0.5% v/v DMSO in total volume of 200 μl per well. The samples were incubated at 37 °C in CO₂ incubators for 2 h, and then 10 μg/ml brefeldin-A was added. The cells were further incubated for 6-8 h.

Gao F., Mallajoysula V., Arunachalam P.S., van der Ploeg K., Manohar M., Röltgen K., Yang F., Wirz O., Hoh R., Haraguchi E., Lee J.Y., Willis R., Ramachandiran V., Li J., Kathuria K.R., Li C., Lee A.S., Shah M.M., Sindher S.B., Gonzalez J., Altman J.D., Wang T.T., Boyd S.D., Pulendran B., Jagannathan P., Nadeau K.C, & Davis M.M. (2023). Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection. Immunity, 56(4), 864-878.e4.

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Tumor-Induced T Cell Metabolism Modulation

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Human peripheral blood T cell subsets (10⁶/ml) were activated with anti-CD3 (clone UCHT1, 2.5 μg/ml) and anti-CD28 (clone CD28.2, 1.25 μg/ml) antibodies (BD Bioscience) for 3–5 days, as indicated in the figure legends. To obtain tumor medium, primary ovarian cancer cells were cultured for 3 days, and subsequently were frozen and thawed 5 times along with the culture medium. The cancer cell culture supernatant was obtained through centrifugation (20,000g, 1 h, 4°C), and used to culture T cells with or without glucose (2 mg/ml, Sigma Aldrich) supplementation for 16–24 h. To obtain normal medium containing different concentrations of glucose, normal medium without glucose (Invitrogen) was supplemented with 1 or 5 mg/ml glucose. Chemical inhibitors of metabolic pathways, Notch signaling or EZH2 were added as indicated in figure legends. Total numbers of viable cells at the endpoint of experiments were 1–2 × 10⁶ cells.

Zhao E., Maj T., Kryczek I., Li W., Wu K., Zhao L., Wei S., Crespo J., Wan S., Vatan L., Szeliga W., Shao I., Wang Y., Liu Y., Varambally S., Chinnaiyan A.M., Welling T.H., Marquez V.E., Kotarski J., Wang H., Wang Z., Zhang Y., Liu R., Wang G, & Zou W. (2015). Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction. Nature immunology, 17(1), 95-103.

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