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Anti cd28 apc

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Anti-CD28 APC is a monoclonal antibody conjugated with allophycocyanin (APC) that binds to the CD28 receptor on the surface of T cells. CD28 is a co-stimulatory molecule that plays a crucial role in T cell activation and proliferation. The APC fluorophore allows for the detection and analysis of CD28-positive cells using flow cytometry.

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Lab products found in correlation

Anti cd4 pe cy7, by BD (2 mentions) Anti il 17a pe, by Thermo Fisher Scientific (2 mentions) Lsrfortessa, by BD (2 mentions) Anti cd3 pacific blue, by BD (1 mentions) Anti cd19 apc, by BD (1 mentions) Anti cd56 fitc, by BD (1 mentions) Anti cd62l pe cy7, by Thermo Fisher Scientific (1 mentions) Anti cd38 percp cy5.5, by BD (1 mentions) Anti cd45ra percp cy5, by Thermo Fisher Scientific (1 mentions) Anti cd4 apc efluor 780, by Thermo Fisher Scientific (1 mentions) Anti cd1d pe, by BD (1 mentions) Facs lysing solution, by BD (1 mentions) Anti cd27 pe, by BD (1 mentions) Recombinant human cx3cl1, by R&D Systems (1 mentions) Anti cd3 pb, by BD (1 mentions) Anti ifn γ fitc, by BD (1 mentions) Anti cd28 pe, by BD (1 mentions) Anti il 17 alexa647, by BioLegend (1 mentions) Anti cd14 apc cy7, by BD (1 mentions) Anti tnf percp cy5, by BioLegend (1 mentions)

8 protocols using anti cd28 apc

1

Multicolor Flow Cytometry Immunophenotyping

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Peripheral blood lymphocytes were immunophenotyped by multicolour flow cytometry using the following antibodies: anti-CD3 Pacific Blue, anti-CD56 FITC, Anti-CD27 PE, anti-CD28 APC, anti-CD1d PE, anti-CD19 APC, anti-CD27 V450, anti-CD38 PerCP-Cy5.5 (all from BD Biosciences, San Jose, CA, USA) and anti-CD45RA PerCP-Cy5.5, anti-CD62L PE-Cy7, anti-CD5 PE-Cy7, anti-CD24 APC-eFluor 780 and anti-CD4 APC-eFluor 780 (from eBioscience, Inc. San Diego, CA, USA). Staining was performed on whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturer’s instructions. A minimum of 250,000 events were acquired for T cell panels and 500,000 events for B cell panels to ensure adequate capture of rare populations. Subsequent detailed analysis of lymphocyte sub-populations was performed on the gated lymphocyte population using FlowJo (Treestar, Inc., OR, USA). Absolute counts for the different lymphocyte populations were calculated per litre of blood, based on haematology laboratory reported total lymphocyte count.
Cooles F.A., Anderson A.E., Drayton T., Harry R.A., Diboll J., Munro L., Thalayasingham N., Östör A.J, & Isaacs J.D. (2016). Immune reconstitution 20 years after treatment with alemtuzumab in a rheumatoid arthritis cohort: implications for lymphocyte depleting therapies. Arthritis Research & Therapy, 18, 302.
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2

Chemotaxis of CD4+ Memory T Cells

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Purified CD4+ memory T cells derived from ESRD patients were stained with anti-CD28-APC (BD Biosciences) for 30 min at 4 °C. Chemotaxis of stained CD4+ memory T cells was analyzed using 24-well Transwell chambers with 5 μM pores (Corning Inc, Corning, NY). 3 × 105 cells in 100 μl chemotaxis buffer (RPMI 1640 with 0.5% BSA) were placed in the upper chambers. Recombinant human CX3CL1 (2.5 to 20 ng/ml; R&D systems) in 600 μl chemotaxis buffer was placed in the lower wells and the chambers were incubated for 2 hours at 37 °C and 5% CO2. Migrated cells located in the bottom wells were collected and analyzed using a BD LSRFortessa.
Kim H.Y., Yoo T.H., Hwang Y., Lee G.H., Kim B., Jang J., Yu H.T., Kim M.C., Cho J.Y., Lee C.J., Kim H.C., Park S, & Lee W.W. (2017). Indoxyl sulfate (IS)-mediated immune dysfunction provokes endothelial damage in patients with end-stage renal disease (ESRD). Scientific Reports, 7, 3057.
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3

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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4

APC Conjugation of Immunomodulatory Proteins

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APC‐conjugated proteins: PD‐1‐Ig (Bio‐Techne, 1086‐PD‐050), anti‐PD‐L1 (Durvalumab) and CTLA‐4 Ig (Abatacept & Belatacept) were generated using the APC Conjugation Kit—Lightning‐Link® according to manufacturer's instructions (Abcam, ab201807). APC‐conjugated anti‐PD‐L1 antibodies were procured from ThermoFisher (clone MIH1 [#14‐5983‐82]), or Biolegend (clones MIH3 [#374513] and 29E.2A3 [#329707]). Anti CD28‐APC was procured from BD Biosciences (#559770).
Kennedy A., Robinson M.A., Hinze C., Waters E., Williams C., Halliday N., Dovedi S, & Sansom D.M. (2023). The CTLA‐4 immune checkpoint protein regulates PD‐L1:PD‐1 interaction via transendocytosis of its ligand CD80. The EMBO Journal, 42(5), e111556.
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5

Phenotypic and Functional Profiling of T Cells

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For suppression assays, cells were washed with 0.1% (w/v) sodium azide/phosphate-buffered saline (Mediatech Cellgro) on day 7 of in vitro stimulation and were stained with anti-CD4 PECy5 (BD Pharmingen), anti-CD8 Pacific Blue (Biolegend), and anti-CD25 APCCy7 (BD Pharmingen. For surface phenotyping of cells, bulk PBMCs and enriched CD8+ T cells were stained with anti-CD3 Alexa 700 (BD Pharmingen), anti-CD8 AmCyan (BD Biosciences), anti-CD27 APCCy7 (Biolegend), anti-CD28 APC (BD Pharmingen), CD45RO Pacific Blue (Biolegend), anti-CD62L PECy5 (BD Pharmingen), and anti-CD57 PE (Southern Biotech). For intracellular staining of cytokines, cells were initially activated with 1 μL of leukocyte activation cocktail (BD Pharmingen) for 5 hours. Cells were surface stained with anti-CD8 APC (BD Biosciences), anti-CD4 PECy5 (BD Pharmingen) and anti-CD25 APCCy7 (BD Pharmingen) and permeabilized as described previously. Intracellular staining was performed using anti-IFNγ PECy7 (BD Pharmingen), anti-IL-17A PE (Ebioscience), anti-Granzyme B Alexa 700 (BD Pharmingen) and anti-Perforin Pacific Blue (BD Pharmingen). All cells were resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfiled, PA) for FACS analysis. Flow cytometric data were acquired on a 4-Laser, 17-color LSRII using FACSDiva software (Becton Dickinson). CFSE was detected in the FITC channel on the LSR.
Cunnusamy K., Baughman E.J., Franco J., Ortega S.B., Sinha S., Chaudhary P., Greenberg B.M., Frohman E.M, & Karandikar N.J. (2014). Disease Exacerbation of Multiple Sclerosis is Characterized by Loss of Terminally Differentiated Autoregulatory CD8+ T cells. Clinical immunology (Orlando, Fla.), 152(0), 115-126.
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6

PBMC Isolation and Flow Cytometry Analysis

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Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden), and then, cryopreserved until use15 (link). Cryopreserved PBMCs were thawed and analysed with flow cytometry. Briefly, cells were stained with fluorochrome-conjugated monoclonal antibodies against surface antigens for 20 min at 4 °C. The antibodies were anti-CD3-Horizon V500, anti-CD4-PE-Cy7, anti-CD8-APC-H7, anti-CD28-APC (all from BD Biosciences) and anti-CD57-Pacific blue (Biolegend, San Diego, CA). Multicolour flow cytometry was performed with an LSR II Flow Cytometer, and data was analysed with FlowJo software.
Youn J.C., Jung M.K., Yu H.T., Kwon J.S., Kwak J.E., Park S.H., Kim I.C., Park M.S., Lee S.K., Choi S.W., Han S., Ryu K.H., Kang S.M, & Shin E.C. (2019). Increased frequency of CD4+CD57+ senescent T cells in patients with newly diagnosed acute heart failure: exploring new pathogenic mechanisms with clinical relevance. Scientific Reports, 9, 12887.
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7

Quantifying T-cell Subsets by Flow Cytometry

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In vitro cultured cells or PBMCs from patient blood samples were washed and stained with a combination of fluorochrome-conjugated monoclonal antibodies. PE-anti-CD4, PE-anti-CD8, APC-anti-CD25, and APC-anti-CD28 antibodies (all from BD Biosciences, San Jose, CA, USA) were used. Labeled cells were incubated away from light for 20 min at room temperature. Next, cells were washed and stained with FITC-conjugated anti-Foxp3 (eBioscience) after fixation and permeabilization according to the manufacturer's instructions. All events were acquired using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using Kaluza 1.3 software (Beckman Coulter).
Wu M., Chen X., Lou J., Zhang S., Zhang X., Huang L., Sun R., Huang P., Wang F, & Pan S. (2016). TGF-β1 contributes to CD8+ Treg induction through p38 MAPK signaling in ovarian cancer microenvironment. Oncotarget, 7(28), 44534-44544.
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8

Phenotypic Profiling of Human PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated from human child blood by density gradient centrifugation (Biocoll Separating Solution; Biochrom, Cambridge, UK). Phenotypic staining was performed on the day of isolation, and the remaining cells were frozen in cryovials in liquid nitrogen until use. Freshly-isolated PBMCs were stained at 4 °C for 30 min with the following fluorochrome-conjugated antibodies: Alexa700-anti-CD4, V500-anti-CD8, APC-anti-CD28, PE-Cy7-anti-CCR7 (all four from BD Biosciences, Franklin Lakes, NJ, USA), APC-Cy7-anti-CD3, FITC-anti-CD45RA, APC-anti-PD-1, FITC-anti-LAG-3, PE-anti-TIM-3 (all five from BioLegend, San Diego, CA, USA), V450-anti-CD57, and PE-anti-CD85j (both from eBioscience, San Diego, CA, USA) antibodies. Stained cells were acquired using Fortessa-X20 or LSRFortessa (BD Biosciences) and analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).
Hong K.T., Kang Y.J., Choi J.Y., Yun Y.J., Chang I.M., Shin H.Y., Kang H.J, & Lee W.W. (2023). Effects of Korean red ginseng on T-cell repopulation after autologous hematopoietic stem cell transplantation in childhood cancer patients. Journal of Ginseng Research, 48(1), 68-76.
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