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Cd28 pe cy7

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The CD28 PE-Cy7 is a flow cytometry reagent that can be used to detect the expression of the CD28 molecule on the surface of cells. CD28 is a co-stimulatory molecule that plays a critical role in the activation and proliferation of T cells. The PE-Cy7 fluorochrome is conjugated to the CD28 antibody, allowing for the specific detection and quantification of CD28-positive cells in a sample using flow cytometry.

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CD28-PE (16 citations)

18 protocols using cd28 pe cy7

1

Multi-Panel Flow Cytometry for Immune Profiling

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The following immune cell subsets were analysed using flow cytometry; T-cell subsets: anti-CD3-BV510 (BD, Cat: 564713, clone HIT3a), CD8-APC-H7 (BD, Cat: 560179, clone SK1), CD45RA-BB515 (BD, Cat: 564552, clone HI100), CCR7-PE (BioLegend, Cat: 353204, clone G043H7), CD28-PeCy7 (BD, Cat: 560684, clone CD28.2), CD137 (4-1BB)-APC (BD, Cat: 550890clone 4B4-1), PD1-BB700 (BD, Cat: 566460, clone EH12.1), intracellular Ki67-BV420 (BD, Cat. 562899, clone B56); Treg cells: anti–CD4-APC-H7 (BD, Cat: 560158, clone RPA-T4); CD25-PE (BioLegend, Cat: 356134, M-A251), CD45RA-BB15 (BD, Cat: 564552, clone HI100), CTLA-4-PerCp-Cy5.5 (BD, Cat: 561717, BNI3); FOXP3OX-APC (Thermo Fisher Scientific, Cat: 17-4776-42; PCH101); MDSCs and DC CD14-BB700 (Biolegend, Cat: 566465, MΦP9); CD66b-PeCy7 (BioLegend, cat: 305116, G101F5); HLA-DR-FITCH (BioLegend, Cat: 307604, L243); CD45-AF700 (BioLegend, Cat: 368514 2D1); CD11c-BV421 (BioLegend, Cat: 301628, 3.9). Cell autofluorescence and fluorescence minus one (FMO) were used as negative controls. Flow cytometric acquisition was performed using a FACSCantoII flow cytometer running FACS Diva data acquisition. FACS DIVA analysis software (version 8.0.2, BD Biosciences) and FlowJo (version 10.8.8, BD) were used to analyse the data.
Scirocchi F., Scagnoli S., Botticelli A., Di Filippo A., Napoletano C., Zizzari I.G., Strigari L., Tomao S., Cortesi E., Rughetti A., Marchetti P, & Nuti M. (2022). Immune effects of CDK4/6 inhibitors in patients with HR+/HER2− metastatic breast cancer: Relief from immunosuppression is associated with clinical response. EBioMedicine, 79, 104010.
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2

Flow Cytometric Immune Profiling in Transplant

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Flow cytometric analysis was performed up to 3 times pre-transplant and serially post-transplant to characterize peripheral blood immune cell phenotypes. Total T cells and T cell subsets were quantified by complete blood cell count and flow cytometry. Fresh PBMCs were isolated by Ficoll density gradient centrifugation (BD Biosciences, Franklin Lakes, NJ). PBMCs were stained with the following mAbs: CD3 PacBlue, CD95 V450, CD3 Alexa 700, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD25 PE-Cy7, IFNy PE-Cy7, CD28 APC, TNF APC, VLA-4 APC, CD11a PE, CD45RA FITC, CD40 FITC, CCR7 APC, CD20 APC (all BD Biosciences). PBMCs (1.5×106) were incubated with appropriately titered antibodies for 15min at 20°C and washed twice. Samples were acquired immediately on a BD LSR II multicolor flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). For the stimulation assay, 1.5×106 PBMCs were cultured in RPMI 1640 (Corning cellgro, Manassas, VA) supplemented with 10% fetal bovine serum and stimulated with 10µM phorbol 12-myristate 13-acetate (PMA) and 200nM ionomycin (Sigma-Aldrich, St. Louis, MO), with 1ul/ml GolgiPlug protein transport inhibitor for 5 h, +/− IL-15 (10ng/mL). PBMCs were were processed with BD Cytofix/Cytoperm Plus kit (BD 555028) per the manufactures recommendation prior to data acquisition.
Mathews D., Wakwe W., Kim S., Lowe M., Breeden C., Roberts M., Farris A., Strobert E., Jenkins J., Larsen C., Ford M., Townsend R, & Adams A. (2017). Belatacept Resistant Rejection is Associated with CD28+ Memory CD8 T cells. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 17(9), 2285-2299.
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3

Comprehensive Immune Cell Profiling Post-Transplantation

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Analysis of circulating immune cell phenotypes was performed both prior to transplant and at regular intervals following transplantation. Cell frequencies from flow cytometric analysis were combined with complete blood counts to calculate total numbers of circulating T cells and various T cell subsets. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (BD Biosciences, Franklin Lakes, NJ) within 6 hours of phlebotomy. PBMCs (1.5×10^6) were then incubated with antibody mixtures at the appropriate titer for 15 minutes and washed twice. For assessment of intracellular markers, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s direction following surface staining. Flow cytometric data was acquired immediately using a BD LSR II multicolor flow cytometer (BD Biosciences). All flow data was analyzed using FlowJo (Tree Star, San Carlos, CA).
Surface markers were stained with the following monoclonal antibodies (mAbs): CD3 PacBlue, CD3 APC-Cy7, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD127 PE-Cy7, PD-1 APC, LFA-1 APC, CD20 APC-Cy7 (all BD Biosciences), CD95 PacBlue, CD69 FITC (Invitrogen, Grand Island, NY), and CD25 PE (Miltenyi Biotech, San Diego, CA). Intracellular staining for FoxP3 was performed using FoxP3 Alexa488 (Biolegend, San Diego, CA).
Anderson D.J., Lo D.J., Leopardi F., Song M., Strobert E.A., Jenkins J.B., Larsen C.P, & Kirk A.D. (2019). Corticosteroids and Methotrexate as Adjuvants to Costimulation Blockade in Nonhuman Primate Renal Transplantation. Clinical transplantation, 33(6), e13568.
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4

Multiparametric Immune Cell Profiling

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PBMCs were labeled with different combinations of the following antibodies: CD4 BUV737 (BD, clone SK3), CD8 BV786 (BD, clone RPA-T8), CD8 BUV805 (BD, clone SK1), CD3 V500 (BD, clone UCHT1), CD3 eVolve605 (eBioscience clone OKT3), CD27 APC-eFluor780 (eBioscience, clone), CD27 BV510 (Biolegend, clone O323), CD45RA BV650 (BD, clone HI100), CD45RA Qdot655 (Invitrogen, clone MEM-56), CD28 PE-Cy7 (BD, clone 28.2), CX3CR1 APC (eBioscience, clone 2A9-1), CCR7 BV421 (Biolegend, clone G043H7), CD127 BV421 (Biolegend, clone A019D5). Near-IR fixable dye (Invitrogen) was used to exclude dead cells from the analysis. For intracellular staining, the following antibodies were used: Hobit IgM (BD, clone Sanquin-Hobit/1), Eomes eFluor660 (eBioscience, clone WD1928), Tbet BV421 (Biolegend, clone 4B10), Granzyme B AF700 (BD, clone GB11), Perforin FITC (eBioscience, clone dG9), Perforin PE (eBioscience, clone B-D48), Granzyme K PE (Immunotools, clone 24C3), Granzyme K FITC (Immunotools, clone 24C3). To stain for Hobit IgM, a secondary anti-IgM labeled with PE or FITC was used. The cells were labeled according to manufacturer’s instructions. For the intracellular staining, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining kit (eBioscience). The samples were measured in PBS 0.5% FCS with a LSR Fortessa (BD). The analysis was done using FlowJo Version 10 software.
Oja A.E., Vieira Braga F.A., Remmerswaal E.B., Kragten N.A., Hertoghs K.M., Zuo J., Moss P.A., van Lier R.A., van Gisbergen K.P, & Hombrink P. (2017). The Transcription Factor Hobit Identifies Human Cytotoxic CD4+ T Cells. Frontiers in Immunology, 8, 325.
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5

MHC Tetramer-Based T-Cell Immunophenotyping

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MHC class I tetramer was prepared in-house. Surface staining of T cells was achieved by addition of tetramer to whole blood, incubation for 10 minutes at room temperature, followed by addition of antibody co-stains for 20 minutes. Whole blood was preferred to thawed PBMCs for tetramer labelling due to greater consistency and signal intensity. Following lysis of erythrocytes using BD FACS Lysing solution (BD Biosciences), cells were either fixed using 2% formaldehyde (v/v) or permeabilized using the BD Cytofix/Cytoperm kit for intra-cellular staining. The following antibodies were used for surface and intra-cellular staining: CD3-PerCP, CD8-Horizon V500, CD8-APCH7, Ki-67-FITC, Bcl-2-PE, HLA-DR-Horizon V450, HLA-DR-PerCP, Perforin-FITC, Granzyme B-Horizon V450, CCR7-PE, CD27-Horizon V450, CD27-FITC, CD28-PECy7, CD28-PE (all BD Biosciences) and CD38-PECy7 (eBioscience), Granzyme B-PE (Caltag), Granzyme K-PE (Santa Cruz), CD45RA-FITC (Beckman Coulter), PD-1-PE and 2B4-PerCPCy5.5 (Biolegend). Intra-cellular cytokine staining with IFN-γ-FITC, TNF-α-APC, and IL-2-PerCpCy5.5 (all BD Biosciences) was undertaken after in vitro stimulation of PBMCs using peptide for 6 hours. Flow cytometry analysis was performed BD LSRII and BD FACSCanto flow cytometers. Flow cytometry data were analyzed using FlowJo software.
Chiu C., McCausland M., Sidney J., Duh F.M., Rouphael N., Mehta A., Mulligan M., Carrington M., Wieland A., Sullivan N.L., Weinberg A., Levin M.J., Pulendran B., Peters B., Sette A, & Ahmed R. (2014). Broadly Reactive Human CD8 T Cells that Recognize an Epitope Conserved between VZV, HSV and EBV. PLoS Pathogens, 10(3), e1004008.
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6

Intracellular Cytokine Production in T and NKT Cells

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To determine whether GCR and SIRT1 are associated with intracellular cytokine production in CD8+ and CD8– T and NKT-like cells, aliquots of blood were stimulated and treated as described above. Following washing of permeabilized cells, appropriately diluted GCR antibody (MCA2469, Bio-Rad, Sydney, Australia) was added to cells for 15 min. Cells were further washed and stained with anti-mouse IgG1 V450 secondary antibody for 15 min. Cells were then washed and stained with SIRT1 Alexa-Fluor 488, IFNγ PE (BD), TNFα PE (BD), CD3 perCP.CY5.5 (BD, Sydney, Australia), CD28 PECY7 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), and CD45 V500 (BD) for 15 min in the dark at room temperature. Appropriate controls and cells were analyzed as above.
Hodge G., Tran H.B., Reynolds P.N., Jersmann H, & Hodge S. (2020). Lymphocyte senescence in COPD is associated with decreased sirtuin 1 expression in steroid resistant pro-inflammatory lymphocytes. Therapeutic Advances in Respiratory Disease, 14, 1753466620905280.
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7

Cytokine and Receptor Expression in T and NKT Cells

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To determine possible association of pro-inflammatory cytokines and Pgp1 and GCR expression by CD28+ and CD28null T and NKT-like cells, whole blood was stimulated as described above. Following stimulation and processing, cells were labeled with anti-GCR as described above, then 5 μL of appropriately diluted IFNγ FITC (BD), TNFα FITC (BD), Pgp1 PE (BD), CD3 perCP.Cy5.5 (BD), CD28 PECY7 (BD), CD56 APC (Beckman Coulter), CD8 APCH7 (BD) and CD45 V500 (BD) were added for 15 min in the dark at room temperature. Cells were washed and events acquired and analyzed as described [5 (link),11 (link),13 ].
Hodge G., Jersmann H., Tran H.B., Holmes M., Reynolds P.N, & Hodge S. (2015). Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes. Respiratory Research, 16(1), 2.
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8

Flow Cytometric Analysis of GCR

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Following stimulation as described above, 350 μL aliquots of cells were treated with 2 mL FACSLyse (BD Bioscience, Sydney, Australia) for 10 min. Cells were centrifuged, supernatant discarded and 500 mL FACSPerm (BD) added for 10 min. Two mL 0 · 5% bovine serum albumin (BSA) (Sigma) in IsoFlow (Beckman Coulter, Sydney, Australia) was then added and the tubes centrifuged at 300 g for 5 min. After decanting supernatant, Fc receptors were blocked with 10 mL human immunoglobulin (Intragam, CSL, Melbourne, Australia) for 10 min at room temperature. Five μL of mouse anti-human GCR (clone 5E4, Serotec, Sydney, Australia; raised against a conserved sequence of the regulatory part of the receptor- amino acids 150–176) as previously reported [16 (link)] was added to cells for 15 min, and following washing (as above), 5 μL rat anti-mouse IgG1 V450 (BD) was added for 15 min. Following washing, 5 μL of appropriately diluted CD3 perCP.Cy5.5 (BD), Pgp1 PE (BD), CD28 PECY7 (BD), CD56 APC (Beckman Coulter), CD8 APCH7 (BD) and CD45 V500 (BD) were added for 15 min in the dark at room temperature. Cells were washed and events acquired and analyzed as previously reported [11 (link),13 ].
Hodge G., Jersmann H., Tran H.B., Holmes M., Reynolds P.N, & Hodge S. (2015). Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes. Respiratory Research, 16(1), 2.
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9

Expanded TIL Immunophenotyping by Flow Cytometry

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Expanded TIL were stained in 100 µL of FACS Wash Buffer (Dulbecco’s Phosphate Buffered Saline 1× with 1% Bovine Serum Albumin) for 30 min using fluorochrome-conjugated monoclonal antibodies for CD3 (FITC), CD16 (PE), γδTCR (APC), CD56 (PE-Cy7), CD4 (PerCP Cy5.5), CD8 (PB), BLTA (PE), CD28 (PE-Cy7), TIM3 (APC) (all BD Bioscience, San Jose, CA), PD-1 (PerCP-Cy5.5) (Biolegend, San Diego, CA) and AQUA live/dead dye (Invitrogen, Carlsbad, CA). Stained cells were acquired using the BD FACScanto II and analyzed using FlowJo software v 10.1 (Tree star, Ashland, OR).
Tavera R.J., Forget M.A., Kim Y.U., Sakellariou-Thompson D., Creasy C.A., Bhatta A., Fulbright O.J., Ramachandran R., Thorsen S.T., Flores E., Wahl A., Gonzalez A.M., Toth C., Wardell S., Mansaray R., Radvanyi L.G., Gombos D.S., Patel S.P., Hwu P., Amaria R.N., Bernatchez C, & Haymaker C. (2018). Utilizing T-cell activation signals 1, 2 and 3 for tumor-infiltrating lymphocytes (TIL) expansion: the advantage over the sole use of interleukin-2 in cutaneous and uveal melanoma. Journal of immunotherapy (Hagerstown, Md. : 1997), 41(9), 399-405.
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10

Multiparameter Flow Cytometry of Immune Cells

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Analysis of circulating immune cell phenotypes was performed both prior to transplant and at regular intervals following transplantation. Data from flow cytometric analysis was combined with complete blood counts to calculate total numbers of circulating T cells and various T cell subsets. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (BD Biosciences, Franklin Lakes, NJ) within 6 hours of phlebotomy. These fresh PBMCs (1.5x10^6) were incubated with antibody mixtures at the appropriate titer for 15 minutes then washed twice. Following surface staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s direction. Flow cytometric data was acquired immediately using a BD LSR II multicolor flow cytometer (BD Biosciences). All flow data was analyzed using FlowJo (Tree Star, San Carlos, CA).
Surface markers were stained with the following monoclonal antibodies (mAbs): CD3 PacBlue, CD3 APC-Cy7, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD127 PE-Cy7, CD11a APC, CD20 APC-Cy7 (all BD Biosciences), CD95 PacBlue, CD69 FITC (Invitrogen, Grand Island, NY), and CD25 PE (Miltenyi Biotech, San Diego, CA). Intracellular staining for FoxP3 was performed using FoxP3 Alexa488 (Biolegend, San Diego, CA).
Anderson D.J., Lo D.J., Leopardi F., Song M., Turgeon N.A., Strobert E.A., Jenkins J.B., Wang R., Reimann K.A., Larsen C.P, & Kirk A.D. (2016). Anti-LFA-1 Therapy in a Nonhuman Primate Renal Transplant Model of Costimulation Blockade Resistant Rejection. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(5), 1456-1464.
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