Anti cd28 pe

Manufactured by BD

Sourced in United States

Anti-CD28-PE is a monoclonal antibody conjugated with the fluorescent dye Phycoerythrin (PE). It binds to the CD28 surface antigen, which is expressed on T cells and plays a role in T cell activation and co-stimulation.

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Anti-CD28 (379 citations) CD28-PE (16 citations) PE-conjugated anti-CD28 (5 citations) PE Mouse Anti-Human CD28 (2 citations)

9 protocols using anti cd28 pe

Multiparametric Flow Cytometry Analysis

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The fluorochrome labeled monoclonal antibodies (mAbs) anti-CD2-FITC, anti-CD3-Alexa 700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-APC Fluor 780, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20 PECy7, anti-CD19-V450, anti-CD20-APCCy7, anti-CD25-PEcy7, anti- CD28-PE, anti-CD28-FITC, anti-CD38-PE, anti-CD39-FITC, anti-CD45-PerCP, anti-CD56-APC, anti-CD57-FITC, anti-CD197-PECy7, anti-HLA-DR, anti-IgM, anti-IgD, anti-Ki67-FITC, anti-Bcl-2-PE, anti-TNF-α-PE, anti-TNF-α-APC, IFN-γ-PcpCy5.5, and IL-2-PECy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-CD45RA-QDOT655 and anti-CD69-FITC mAb were obtained from Invitrogen (Carlsbad, CA). Anti-CD8-Alexa780, anti-CD27-Alexa Fluor 700, and anti-CD38-eFluor 650NC were obtained from E-bioscience (San Diego, CA). Anti-CD24-PEcy7 and anti-CD279-PE (PD-1) were purchased from Biolegend (San Diego, CA).
Human EBV protein and CMV peptide pool of pp65 sequence consisting of 138 peptides (15 mers with 11 amino acid overlaps) was purchased from JPT Peptide Technologies (Berlin, Germany).
All patients were DSA-free with a calculated panel reactive antibody (PRA) ≤20% at enrollment. Patient samples were assessed for donor-specific alloantibody post-transplantation as described previously ^{(18 (link))}.

Xu H., Samy K.P., Guasch A., Mead S.I., Ghali A., Mehta A., Stempora L, & Kirk A.D. (2015). Post Depletional Lymphocyte Reconstitution During Belatacept and Rapamycin Treatment in Kidney Transplant Recipients. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(2), 550-564.

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Evaluating In Vitro Binding Capacity of Immune Checkpoint Proteins

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Stable cell lines were generated to evaluate in vitro binding capacity of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins. For this purpose, total RNA was extracted from anti-CD3/CD28 activated human PBMCs, and the first-strand cDNA was synthesized by Thermo cDNA synthesis kit (Thermo Scientific, USA). Human PD-1 (hPD-1), CTLA-4 (hCTLA-4), CD28 (hCD28), and 4-1BB (h4-1BB) coding genes were amplified by PCR using specific primers and separately inserted into pCHO1.0 expression vector. As described earlier, these constructs were transfected to CHO-K1 by electroporation, and then stable cell lines were produced by puromycin/MTX strategy. The expression of these proteins on the surface of transfected CHO-K1 cells was screened by flow cytometry using anti-PD-1-PE (BioLegend™, USA), anti-CD28-PE (BD Pharmingen™, USA), anti-CTLA4-PE (BioLegend), and anti-4-1BB-PE (BioLegend™, USA) antibodies.

Kaviani E., Hosseini A., Mahmoudi Maymand E., Ramzi M., Ghaderi A, & Ramezani A. (2022). Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities. PLOS ONE, 17(12), e0275777.

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Multi-Marker Phenotyping of T Cells

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Anti-CD4 PerCP, Anti-CD4 PerCP-cy5.5, anti-CD3 APC, anti-CXCR5-Alexafluor 488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, anti-IL-21 PE, anti-IL-4 PE, anti-IFN-γ APC and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-17 APC and ELISA for determining IL-21 was purchased from eBioscience (San Diego, CA, USA). Anti-IL-22 PE was purchased from R & D Systems (Abingdon, UK). PMA, ionomycin (INO), saponin and Brefeldin A were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

Zhou M., Zou R., Gan H., Liang Z., Li F., Lin T., Luo Y., Cai X., He F, & Shen E. (2014). The effect of aging on the frequency, phenotype and cytokine production of human blood CD4 + CXCR5 + T follicular helper cells: comparison of aged and young subjects. Immunity & Ageing : I & A, 11, 12.

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Cytokine Production in Activated T Cells

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Cells were cultured for 6h in complete media (RPMI, HEPES, L-Glutamine, Penicillin, Streptomycin) containing 5% human serum and stimulated with plate-bound α-CD3 monoclonal antibody (plate coated with 2.5 μg/ml, clone OKT-3) and incubated at 37°C, 5% CO₂. In order to prevent produced cytokines from being secreted, 10 μg/ml Brefeldin A (Sigma-Aldrich, Steinheim, Germany) was added to the cultures 4h prior to harvesting. Extracellular and intracellular cytokine staining was performed using Cytofix/Cytoperm Kit (BD) according to the manufacturer’s instructions. Antibodies: Anti-CD28 PE or anti-CD28 APC, anti-CD14 APC Cy7, anti-CD4 PeCy7 or anti-CD4 PerCP, anti-IFN-γ FITC (all BD), anti-CD3 PB (BD or Biolegend), anti-TNF PerCP Cy5.5 (Biolegend), anti-IL17 Alexa 647 (Biolegend) or anti-IL17A PE (eBioscience). LIVE/DEAD Aqua Dead Cell Stain (Invitrogen) was used to exclude dead cells. The PBMCs were run on a Beckman Coulter CyAn. Analyses were performed with FlowJo software, version 8.1.0 or higher (Treestar Inc.).

Pieper J., Johansson S., Snir O., Linton L., Rieck M., Buckner J.H., Winqvist O., van Vollenhoven R, & Malmström V. (2014). Peripheral and site-specific CD4+CD28null T cells from Rheumatoid Arthritis patients show distinct characteristics. Scandinavian journal of immunology, 79(2), 149-155.

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Multicolor Flow Cytometry Panels

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The following flow cytometry antibodies were purchased from BD Biosciences (San Jose, CA): anti-CD3-PE Cy7, anti-CD4-Alexa488, anti-CD8-PacBlu, anti-CD28-PE, anti-CD57-APC, anti-CD45RO-Alexa 700, anti-HLA-DR-APC, anti-CD14-PacBlu, anti-CD11a-FITC, anti-CD16-PE-Cy7, anti-CD163-PE, anti-CD62L-APC, and anti-CD86-Alexa 700. Two multi-color panels were used for T cells, and two panels were used for monocytes. Data were collected using a BD LSRII flow cytometer and analyzed with FCS Express software (DeNovo Software, Los Angeles, CA).

Wallet M.A., Buford T.W., Joseph A.M., Sankuratri M., Leeuwenburgh C., Pahor M., Manini T., Sleasman J.W, & Goodenow M.M. (2015). Increased inflammation but similar physical composition and function in older-aged, HIV-1 infected subjects. BMC Immunology, 16, 43.

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Comprehensive Immune Profiling of Whole Blood

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One hundred μL of whole blood was incubated for 30 min at room temperature with optimized doses and combinations of the following anti-human antibodies: anti-CD3-PerCP (SK7, BD, San Jose, CA) or anti-CD3-Alexa Fluor 700 (UCHT1, Thermo Fisher, Waltham, MA, USA), anti-CD8-PE (RPA-T8; BD) or anti-CD8-Alexa Fluor 700 (OKT8; Thermo Fisher), anti-CD4-APC-eFluor 780 (RPA-T4, Thermo Fisher), anti-HLA-DR-FITC or APC-eFluor 780 (LN3, Thermo Fisher), anti-CD38-PE Cy7 or PE-eFluor 610 (HIT2, Thermo Fisher), anti-PD-1-PerCP-eFluor 710 (F38-2E2, Thermo Fisher), anti-CCR7-PE (3D12, Thermo Fisher), anti-CD45RO-PE (UCHL1, BD), anti-CD45RA-PE Cy7 (HI100, BD), anti-CD28-PE (L293, BD), anti-CD57-FITC (TB01, Thermo Fisher), anti-CD95-FITC (DX2, BD) and anti-CD152-PE (BNI3, BD Pharmingen). Next, red blood cells were removed with 1X FACS Lysing Solution (BD) during 20 min at room temperature, followed by a washing step with 1 mL of 1X PBS and fixation in 1% paraformaldehyde. The cells were acquired on a LSR Fortessa cytometer (BD), using the FACS Diva software v. 6.0, within one hour of completing the staining; at least 50,000 CD3⁺ events were acquired. Data were analyzed with the FlowJo Software version 10.4 (Tree Star, Inc, Ashland, OR, USA). Fluorescence minus one control was included to define positive thresholds.

Perdomo-Celis F., Velilla P.A., Taborda N.A, & Rugeles M.T. (2019). An altered cytotoxic program of CD8+ T-cells in HIV-infected patients despite HAART-induced viral suppression. PLoS ONE, 14(1), e0210540.

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PBMC Cryopreservation and Cell Sorting

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Cryopreserved PBMCs were thawed, washed, counted, and stained for sorting as previously described (39 (link)). Before staining, an aliquot of 1 million PBMCs was removed, pelleted, and frozen at −80 °C to assess PBMC TL. Thawed cells were first stained with LIVE/DEAD^™ Fixable Aqua Dead Cell Stain Kit (Invitrogen) for exclusion of non-viable cells, and were then stained with the following fluorescently conjugated monoclonal antibodies: anti-CD4-PE-Texas Red^® (Invitrogen); anti-CD3-V450, anti-CD19 PE-CyTM5, anti-CD28-PE, anti-CD45 FITC and anti-CD8-APC (all from BD Biosciences). Stained cells were sorted on a customized BD FACSAriaTM II into the following fractions, using standard gating strategies: CD4+ T-cells (CD45+CD3+CD4+), CD8+ T-cells (CD45+CD3+CD8+), and CD19+ B-cells (CD45+CD3-CD19+). Cells were collected into AIM V serum-free media (Invitrogen), pelleted by centrifugation, and stored at −80 °C.

Mason A.E., Hecht F.M., Daubenmier J.J., Sbarra D.A., Lin J., Moran P.J., Schleicher S.G., Acree M., Prather A.A, & Epel E.S. (2018). Weight loss, weight-loss maintenance, and cellular aging in the Supporting Health through Nutrition and Exercise (SHINE) Study. Psychosomatic medicine, 80(7), 609-619.

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T Cell Subset Isolation and Characterization

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T cells were isolated by flow cytometry on a MoFlo (Beckman Coulter) by sorting for CD3⁺CD4⁺ and either CD28⁺ or CD28⁻. The purity of isolated subsets was on average 98%.
All surface staining was carried out in PBS supplemented with 1% human sera and 0.05 % sodium azide using anti-CD3 PC5 (Beckman Coulter) or anti-CD3 FITC, anti-CD4 FITC or anti-CD4 PC5 or anti-CD4 APCCY7, CD45RO PC5 and anti-CD28 PE (all BD) antibody conjugates.

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Phenotypic Analysis of Activated T Cells

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OKT-CIK and R-CIK cells were measured on the 10th and 16th days after RetroNectin and OKT3 stimulating. The cells were harvested and washed in cold PBS and resuspended at 1 × 10⁶ in 100 μL cold PBS consisting of 5% serum. The cells were stained for anti-CD3-APC, anti-CD4-PerCP, anti-CD8-PerCP, anti-CD27-FITC, anti-CD28-PE, anti-CD56-PE, and anti-PD-1-PE purchased from BD Biosciences (San Jose, CA, USA) for 20 minutes on ice. FACS analysis was performed by using a FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA, USA).

Han L., Shang Y.M., Song Y.P, & Gao Q.L. (2016). Biological Character of RetroNectin Activated Cytokine-Induced Killer Cells. Journal of Immunology Research, 2016, 5706814.

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