Fresh PBMCs were stained with anti-CD3-APC-Cy7, CD8-APC, CD45RA-PE-Cy5, CCR7-PE-Cy7, PD-1-Pacific Blue, IL-7Rα-FITC, CD27-PE, CD28-PE, CTLA4-PE, CX3CR1-PE antibodies or isotype control (all from BioLegend, San Diego, CA). PBMCs that had been stimulated with PMA/ionomycin were stained with anti-CD3-APC-Cy7, CD8-APC, CD45RAPE-Cy5 and CCR7-PE-Cy7 antibodies followed by fixation, permeabilization (Cytofix/Cytoperm Kit, BD Biosciences) and staining with anti-IFNγ-PE, TNF-α-FITC or IL-13-PE antibodies (BioLegend).14 (link) Stained cells were analyzed on an LSRII® flow cytometer (BD Biosciences). Collected data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Cd28 pe
Manufactured by BioLegend
Sourced in United States
CD28-PE is a fluorescently labeled monoclonal antibody that binds to the CD28 cell surface receptor. CD28 is a co-stimulatory molecule expressed on T cells that plays a critical role in T cell activation and proliferation. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD28-expressing cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by BD (1 mentions)
Ccr7 pe cy7, by BD (1 mentions)
Anti cd3 apc cy7, by BD (1 mentions)
Cd8 apc, by BD (1 mentions)
Cd8 apc, by BioLegend (1 mentions)
Tnf α fitc, by BioLegend (1 mentions)
Ccr7 pe cy7, by BioLegend (1 mentions)
Anti ifn γ pe, by BioLegend (1 mentions)
Cd45ra pe cy5, by BD (1 mentions)
Anti cd3 apc cy7, by BioLegend (1 mentions)
Cx3cr1 pe, by BioLegend (1 mentions)
Cd27 pe, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cd45ra pe cy5, by BioLegend (1 mentions)
Pd 1 pacific blue, by BioLegend (1 mentions)
Il 13 pe, by BioLegend (1 mentions)
Ctla 4 pe, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Ccr7 pe, by BioLegend (1 mentions)
4 protocols using cd28 pe
1
Comprehensive Immune Cell Profiling
2
Comprehensive T-cell Immunophenotyping Protocol
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Flow cytometry antibodies used for surface marker staining are listed as follows. Antibodies from BD Biosciences include: CD3‐BUV395 (Clone UCHT1; cat #563 546), CD4‐BV786 (Clone SK3; cat #563 877) and CD8‐PE‐Cy7 (Clone SK1; cat #335 787). Antibodies from Biolegend include: CD3‐FITC (Clone UCHT1; cat #300 406), CCR7‐PE (Clone G043H7; cat #353 204), CD45RA‐APC (Clone HI100; cat #304 112), CD45RO‐Pacific Blue (Clone UCHL1; cat #304 215),CD27‐Pacific Blue (Clone M‐T271; cat #356 413), CD28‐FITC (Clone CD28.2; cat #302 906), CD28‐PE (Clone CD28.2; cat #302 907), CD95‐FITC (Clone DX2; cat #305 605), CD127‐Brilliant Violet 785 (Clone A019D5; cat #351 329), TIM‐3‐Pacific Blue (Clone F38‐2E2; cat #345 041), CD57‐FITC (Clone HNK‐1; cat #359 603), LAG‐3‐Brilliant Violet 785 (Clone 11C3C65; cat #369 321) and human TruStain FcX reagent (Cat #422 302). Antibodies for intracellular staining include granzyme B‐PE (BD Biosciences, GB11; cat #561 142), IFN‐γ‐FITC (Miltenyi Biotec, cat #130‐090‐433), IL‐2‐PE (Miltenyi Biotec, cat #130‐090‐487) and TNF‐α‐APC (Miltenyi Biotec, cat #130‐091‐267).
3
Intracellular Cytokine Profiling of T Cells and NK Cells
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To measure the levels of intracellular cytokines of CD4+ and CD8+ T cells as well as NK cells, PBMCs (1×106 cells/mL) were cultured for 4 hours with and without 2 μl Cell Stimulation Cocktail [containing PMA (40.5 μM), ionomycin (669.3 μM), and Brefeldin A (2.5 mg/ml)] (Biolegend, San Diego, USA). Following cell culture, PBMCs were washed with PBS and supernatants were discarded. Prior to the detection of intracellular cytokine levels, cell surface staining was performed using anti-CD3-BV785, -CD4-BV510, -CD8-PE/Cy5, -CD28-PE, -CD56-FITC and -CD57-BV711 mAbs (all from Biolegend, San Diego, USA). For intracellular staining, Fix&Cell Permeabilization Kit (Invitrogen, California, USA) was used according to the manufacturer’s protocol. Briefly, cells were fixed and then permeabilized together with addition of anti-IFN-γ-PE/Cy7, -TNF-α-APC/Cy7 and -IL-10-BV421 mAbs. Stained PBMCs were washed after the incubation and samples were analyzed on a NovoCyte flow cytometer running NovoExpress software (Agilent Technologies, USA).
4
Comprehensive Immune Cell Profiling
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PBMCs thawed on day one were washed twice in PBS + 1% BSA (PBS/BSA) and resuspended in 100µL PBS/BSA containing previously titrated combinations or singles of fluorescently conjugated antibodies against human 1 µg/mL CD3 Pacific Blue (Clone UCHT1; BD Bioscience), 1 µg/mL CD4 FITC (Clone OKT4; BioLegend), 0.2 µg/mL CD28 PE (Clone CD28.2; BioLegend), 1.5 µg/mL CCR7 APC (Clone G043H7; BioLegend), 0.5 µg/mL CD45RO APC-Cy7 (Clone UCHL1; BioLegend), 0.5 µg/mL CD45RA PE-CY7 (Clone HI100; BioLegend), 0.1 µg/mL CD56 PE (Clone HCD56; BioLegend), 1.5 µg/mL CD19 APC-Cy7 (Clone HIB19; BioLegend), and 5µL CD16 FITC (Clone B73.1; BD Biosciences). Cells were analyzed separately for T cells (CD3, CD4, CD28, CD45RA, CD45RO, and CCR7), NK cells (CD3, CD56, and CD16), and B cells (CD3 and CD19). Cells were incubated with antibodies for 30 min on ice in the dark before washing once in PBS/BSA and fixed with 1% formalin (Sigma Aldrich) for 20 min at RT in the dark. Cells were then washed and resuspended in 300 µL PBS/BSA and immediately analyzed on a BD FACS Canto II equipped with 3 lasers at the Duke Cancer Institute Flow Cytometry Core. All analyses were completed after acquisition using FCS Express v6 (DeNovo Software, CA).
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