T cell phenotype was assessed by flow cytometry (LSRFortessa, BD) using fluorochrome-conjugated antibodies specific for CD3 (clone UCHT1; BD), CD4 (SK3; BD), CD8 (SK1; BD), CD45RO (UCHL1; BD), CX3CR1 (2A9-1; BioLegend), CD57 (HNK-1; BioLegend), CD27 (M-T271; BD), CD28 (CD28.2; BioLegend), CD69 (FN50; BD), and CCR7 (3D12; BD). Viable cells were gated using Live/Dead Aqua viability dye (Invitrogen). For induction and detection of intracellular cytokines, cells were cultured overnight with 20ng/ml recombinant human IL-15 (247-ILB; R&D Systems) or medium control (RPMI 1640 [Gibco], supplemented with 10% fetal bovine serum [Gemini Bio-Products], 1% L-glutamine [Gibco], and 1% penicillin/streptomycin [Gibco]), then treated with brefeldin A (GolgiPlug, BD) for 6h prior to harvest. After Live/Dead and surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD) for 20 min on ice and stained for 40 minutes on ice with anti-IFNγ (B27, BD) and anti-TNF (MAb11, BD). For intracellular accumulation of cytolytic molecules, cells were treated with IL-15-supplemented or control medium for 48 hours, then harvested, stained with Live/Dead and surface antibodies, treated with Cytofix/Cytoperm, stained with anti-granzyme B (GB11; BD) and anti-perforin (B-D48; BioLegend).
Cd27 m t271
Manufactured by BD
CD27 (M-T271) is a laboratory equipment product manufactured by BD. It is a cell surface glycoprotein that functions as a costimulatory molecule during T-cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Golgiplug, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
247 ilb, by R&D Systems (1 mentions)
Cd8 sk1, by BD (1 mentions)
Aqua live dead viability dye, by Thermo Fisher Scientific (1 mentions)
Cd4 sk3, by BD (1 mentions)
Anti ifnγ b27, by BD (1 mentions)
Cd69 fn50, by BD (1 mentions)
Cd45ro uchl1, by BD (1 mentions)
Cx3cr1 2a9 1, by BioLegend (1 mentions)
Cd57 hnk 1, by BioLegend (1 mentions)
Ccr7 3d12, by BD (1 mentions)
Anti granzyme b gb11, by BD (1 mentions)
Cd28 cd28.2, by BioLegend (1 mentions)
Cd3 clone ucht1, by BD (1 mentions)
4 protocols using cd27 m t271
1
Multiparametric Flow Cytometry for T Cell Phenotyping
2
Flow Cytometric Analysis of PBMCs
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Peripheral blood mononuclear cells were obtained using a Ficoll-density gradient. These cells were then stimulated with 50 ng/mL of phorbol 12-myristate 13-acetate, 1 μg/mL of ionomycin, and GolgiStop (BD Biosciences) for 4 hours. The Fc receptor blocking reagent (eBioscience) was initially used in all of the samples, and then sequential surface and intracellular staining were performed with the following antibodies: CD19 (HIB19; BioLegend), CD27 (M-T271; BD Biosciences), BAFF (1D6; eBioscience), and APRIL (REA347; Miltenyi Biotec). A fixable viability stain was applied to exclude dead cells (BD Biosciences). Data were acquired with an LSR Fortessa (BD Biosciences) and were analyzed with FlowJo software (FlowJo LLC).
3
Multiparametric Analysis of Immune Cell Function
4
Comprehensive PBMC Phenotyping by Flow Cytometry
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PBMCs were thawed at 37C into complete RPMI media (with 2% Human AB serum, Gemini Bio), washed with PBS and stained for viability (eFluor 506 viability dye, Invitrogen). 5x106 cells from each sample were subjected to surface stain for lineage and maturation markers followed by staining for intracellular proteins GzmB, Perf, and FoxP3. For surface staining, the cells were resuspended in FcR block (30% in MACS buffer for 15’ at 4C), and then incubated (40’ at 4C) with the panel of surface antibodies that included CD19 (LT19, Miltenyi Biotec), antibodies from Biolegend: CD3 (SK7), CD45RA (HI100), CD127 (A019D5), CD56 (NC1M16.2), CD20 (2H7), and LAP (TW4-6H10); antibodies from BD Bioscience: CD4 (SK3), CD8 (SK1), and CD27 (M-T271). After washing with MACs Buffer (0.1%FBS/PBS, 4C), cells were fixed and permeabilized using the eBioScience FoxP3 fixation buffer set, then incubated with permeabilization buffer containing 10% NRS (normal rat serum, 10’ at 4C), followed by incubation (30’ at 4C) with a panel of intracellular antibodies that included antibodies from Biolegend: Ki67 (KI67), FoxP3 (206D), IFNg (4S.B3), IL-17 (BL168), IL-10 (JES3-9D7) and Perf (dG9), and GzmB (GB11, BD Bioscience). The samples were washed with MACS buffer and each entire sample analyzed on a BD FACS Symphony flow cytometer with HTS attachment.
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