Anti cd28 fitc

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Anti-CD28-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD28 receptor expressed on the surface of T cells. It is used as a tool in flow cytometry and other immunological applications for the identification and analysis of CD28-positive cells.

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Anti-CD28 (379 citations)

8 protocols using anti cd28 fitc

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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Isolation and Expansion of CMV-specific CD4+ T Cells

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Cryopreserved PBMC from CMV-seropositive healthy blood donors were thawed and cultured in the presence of UV-inactivated CMV antigen as previously described^{26 (link)}. The antigen, derived from CMV strain AD 169 grown and in human lung fibroblasts tissue culture, was tittered in optimization assays to yield maximum lymphocyte proliferation in the conditions used in this report. The final concentration of antigen in the assays corresponded to approximately 5*10⁴ plaque forming units/ml. Mock-infected control was processed similar to the CMV-antigen but without the viral infection step. After six days of stimulation, PBMC were washed with 2% fetal bovine serum (FBS) in PBS and stained with anti-CD4 APC (BD Biosciences, clone RPA-T4), anti-CD27 PE (BD Biosciences, clone M-T271), anti-CD28-FITC (BD Biosciences, clone CD28.2) for 30 minutes in the dark. After incubation, PBMC were washed with 2% FBS in PBS and CD4+CD27-CD28- cells were separated using an Astrios cell sorter (Beckman Coulter). Cell purity was ≥98%. Sorted CD4+CD27-CD28- cells were cultured with autologous PBMC in the presence of CMV antigen for six days.

Tovar-Salazar A, & Weinberg A. (2020). UNDERSTANDING THE MECHANISM OF ACTION OF CYTOMEGALOVIRUS-INDUCED REGULATORY T CELLS. Virology, 547, 1-6.

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Comprehensive Immune Cell Profiling

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The following mAbs were used for flow cytometry: anti-CD3 PerCP, anti-CD4 FITC, anti-CD4 PerCP, anti-CD4 PE-Cy7, anti-CD8 PErCP, anti-CD8 APC-Cy7, anti-CD28 FITC, anti-CD95 APC, anti-CD195 (anti-CCR5) PE, anti-Ki67 PE, anti-HLA-DR APC, and anti-HLA-DR FITC (BD Biosciences). Additionally, annexin V PE was purchased from BD Biosciences, anti-CD8 APC and anti-CD8 PE were obtained from Beckman Coulter (CA, USA), and anti-CD38 FITC was purchased from StemCell Technologies (Vancouver, Canada). Flow cytometry data were acquired on a FACSAria II (BD Biosciences). All data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Zhan X.Y., Wang N., Liu G., Qin L., Xu W., Zhao S., Qin L, & Chen X. (2014). Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy. Retrovirology, 11, 112.

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PBMC IFNγ ELISPOT Assay with Peptide Stimulation

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PBMCs responding to both NAE and AE in an IFNγ ELISPOT assay were pulsed with the peptides at 10μM in the presence of anti-CD28 and anti-CD49d. Monensin and brefeldin A were added 1 hour after peptide stimulation. The cells were incubated for an additional 11h. Following incubation, cells were surface stained for 30min at 4°C with dead cell dye (Invitrogen), anti-CD3-Alexa 780 (eBioscience), anti-CD4-Qdot655 (Invitrogen), and anti-CD8-V500 (BD Pharmingen) in the following panels: (1) anti-TIGIT-Percp/CY5.5 (Biolegend), anti-CD160-Alexa488 (eBioscience), anti-PD1-Alexa700 (Biolegend), anti-TIM3-BV421 (Biolegend) and anti-LAG3-PECy7 (Biolegend) and (2) anti-CD28-FITC (BD Pharmingen), anti-CD27-PECy7, anti-CD38-v450 (eBioscience), anti-CD57- Percp/CY5.5 (Biolegend), and anti-CD69-Alexa700 (Biolegend). The cells were then permeabilized and stained with anti-IFNγ-PE at 4°C for 30 min. At least 10⁶ total events were acquired on an LSR II flow cytometer (BD Immunocytometry Systems), and analyzed using FlowJo (version 9.6.4; TreeStar Inc.). The criteria of positivity is the same as defined above for tetramer staining [73 (link)].

Qin K., Boppana S., Du V.Y., Carlson J.M., Yue L., Dilernia D.A., Hunter E., Mailliard R.B., Mallal S.A., Bansal A, & Goepfert P.A. (2019). CD8 T cells targeting adapted epitopes in chronic HIV infection promote dendritic cell maturation and CD4 T cell trans-infection. PLoS Pathogens, 15(8), e1007970.

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Expansion and Characterization of CD90.1+ T Cells

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CD90.1⁺CD4⁺ and CD90.1⁺CD8⁺ sorted T cells were put back in culture for 9 d in the presence of 300 IU/ml human IL-2 (Novartis Proleukin). T cells were then distributed in 96-well plates (10³ cells per well) in 200 µl RPMI 1640 (31870-025; Gibco) supplemented with irradiated (35 Gy) pooled allogenic feeder cells (corresponding to 10⁷ PBMCs from a pool of three donors and 10⁶ cells corresponding to a 1-to-1 mix of two B-lymphoblastoid cell lines established from distinct donors), 1 µg/ml leukoagglutinin (PHA-L; 4144-5MG; Sigma-Aldrich), 7% pooled human serum, GlutaMAX (35050-038; Gibco), penicillin-streptomycin (15140-122; Gibco), and IL-2 (300 IU/ml) as described (Vivien et al., 2018 (link)). After 7 d of culture in 96-well plates, T cells were expanded in RPMI 1640 supplemented with 7% pooled human serum, GlutaMAX, penicillin/streptomycin, and 300 IU/ml human IL-2 for an additional 10 d, a time point when T cells stopped proliferating and reached numbers appropriate for AP-MS analysis. Before use in AP-MS analysis, T cells were analyzed for the expression of mouse CD90.1, and human CD3 and CD28 using anti–CD90.1-BV605 (740373; BD Biosciences), anti–CD3-APC (17-0037-41; Invitrogen), and anti–CD28-FITC (302906; BD Biosciences) antibodies. Control T cells were grown in parallel under the same conditions.

Nicolas P., Ollier J., Mori D., Voisinne G., Celis-Gutierrez J., Gregoire C., Perroteau J., Vivien R., Camus M., Burlet-Schiltz O., Gonzalez de Peredo A., Clémenceau B., Roncagalli R., Vié H, & Malissen B. (2022). Systems-level conservation of the proximal TCR signaling network of mice and humans. The Journal of Experimental Medicine, 219(2), e20211295.

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Multiparametric Flow Cytometry Analysis of T Cell Responses

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The fluorochrome labeled monoclonal antibodies (mAbs) anti-CD3-Alexa 700, anti-CD4-V450, anti-CD4-PEcy7, anti-CD8-APC, anti-CD8-V450, anti-CD2-APC, anti-CD28-FITC, anti-TNF-α-PE, anti-IFN-γ-PerCPcy5.5, and anti-IL-2-PEcy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-CD45RA-QDOT655 mAb and carboxyfluorescein succinimidyl ester (CFSE) were obtained from Invitrogen (Carlsbad, CA). Anti-CD197-APC anti-CD8-Alexa780 mAb was obtained from Ebioscience (San Diego, CA). Human 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). Perm/Wash buffer, Cytofix/Cytoperm solution, and GolgiPlug containing brefeldin A were obtained from BD Biosciences.

Xu H., Perez S.D., Cheeseman J., Mehta A.K, & Kirk A.D. (2014). The allo- and viral-specific immunosuppressive effect of belatacept, but not tacrolimus, attenuates with progressive T cell maturation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14(2), 319-332.

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Multicolor Flow Cytometry Analysis

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CY5-anti-CD3, FITC-anti-CD8, CY5-anti-CD8, CY5-anti-CD4, PE-anti-CD158b (KIR2DS2), PE-anti-NKB1 (KIR3DL1) and FITC-anti-CD28 were obtained from BD PharMingen, San Diego, CA. PE-anti-CD158d (KIR2DL4) was obtained from R&D Systems (Minneapolis, MN) and PE-anti-CD158a/h (KIR2DL1/2DS2) and PE-anti-CD158b1/b2 (KIR 2DL2/2DL3/2DS2) were obtained from Beckman Coulter Immunotech (Fullerton, CA). Five μl of each PE-conjugated anti-KIR Ab were mixed to form a “cocktail” and used to stain the cells (19 (link)). All labeling procedures were performed on ice in PBS containing 10% horse serum and normal human AB serum (Invitrogen) and sodium azide. Cell staining was performed as previously described (20 (link)), and the stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Li Y., Gorelik G., Strickland F.M, & Richardson B.C. (2014). Oxidative Stress, T Cell DNA Methylation, and Lupus. Arthritis & Rheumatology (Hoboken, N.j.), 66(6), 1574-1582.

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Multiparametric Flow Cytometry Panel

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Fluorescein isothiocyanate (FITC) anti-CD3 (SK7); FITC anti-CD19 (HIB19); FITC anti-CD28 (CD28.2); FITC anti-CD45RO (UCHL1); FITC anti-CD56 (NCAM16.2); FITC anti-CD69 (FN50); FITC anti-CD94 (HP-3D9); FITC anti-CD95 (DX2); FITC anti-TCR αβ (WT31); phycoerythrin (PE) anti-CD3 (SK7); PE anti-CD25 (M-A251); PE anti-CD45RA (HI100); PE anti-CD56 (NCAM6,2), PE anti-IL-2 (MQ1-17H12); 7-Amino-Actinomycin D (7-AAD); PE-Cy5 anti-CD3 (UCHT1); PE-Cy7 anti-CD3 (SK7); PE-Cy7 anti-CCR7 (3D12); PE-Cy7 anti-IFNγ (B27); allophycocyanin (APC) anti-CD3 (SK7); APC anti-CD4 (RPA-T4); APC anti-CD8 (RPA-T8); APC anti-CD27 (L128); APC anti-CD45RO (UCHL1); APC-Cy7 anti-CD8 (SK1); Alexa Fluor 700 anti-CD4 (RPA-T4); V450 anti-CD3 (UCHT1) were purchased from BD Biosciences, San Jose, CA, USA. FITC anti-FOXP3 (236A/E7); APC anti-FOXP3 (236A/E7) and Alexa Fluor 700 anti-CD4 (OKT-4) came from eBioscience, Inc., San Diego, CA, USA. Qdot605 anti-CD3 (UCHT1) and Pacific Orange anti-CD8 (3B5) came from Invitrogen, Eugene, OR, USA. FITC anti-TCR PANγδ (IMMU510) and Krome Orange anti-CD4 (13B8.2) was purchased from Beckman Coulter, Immunotech, Marseille, France.

Stikvoort A., Sundin M., Uzunel M., Gertow J., Sundberg B., Schaffer M., Mattsson J, & Uhlin M. (2016). Long-Term Stable Mixed Chimerism after Hematopoietic Stem Cell Transplantation in Patients with Non-Malignant Disease, Shall We Be Tolerant?. PLoS ONE, 11(5), e0154737.

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