Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).
Cd27 m t271
Manufactured by BioLegend
Sourced in United States
CD27 (M-T271) is a monoclonal antibody that recognizes the human CD27 antigen. CD27 is a co-stimulatory molecule that belongs to the tumor necrosis factor receptor superfamily and is expressed on the surface of T cells, B cells, and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Pd 1 eh12.2h7, by BioLegend (1 mentions)
Cd14 m5e2, by BioLegend (1 mentions)
Cd4 okt4, by BioLegend (1 mentions)
Hcd56, by BioLegend (1 mentions)
Cd57 hnk 1, by BioLegend (1 mentions)
Ia6 2, by BioLegend (1 mentions)
Cd20 2h7, by BioLegend (1 mentions)
Cd11c 3, by Thermo Fisher Scientific (1 mentions)
Klrg1 sa231a2, by BioLegend (1 mentions)
Cd86 it2, by BioLegend (1 mentions)
Cd8b 2st8.5h7, by Beckman Coulter (1 mentions)
Sa231a2, by BioLegend (1 mentions)
Cd123 6h6, by BioLegend (1 mentions)
Cd16 3g8, by BioLegend (1 mentions)
Hla dr l243, by BioLegend (1 mentions)
Cd19 hib19, by BioLegend (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus gradient, by GE Healthcare (1 mentions)
Igd ia6 2, by BioLegend (1 mentions)
Foxp3 pch101, by Thermo Fisher Scientific (1 mentions)
4 protocols using cd27 m t271
1
Flow Cytometric Profiling of PBMCs
2
Immunophenotyping of PBMC Subsets
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Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll-Paque PLUS gradient (GE Healthcare; IL, USA). Anti-human antibodies for surface and intracellular makers with isotype-matched controls were used for staining CD3 (UCHT1), CD4 (OKT4), CD8 (RPA-T8), CD45RA (HI100), CD45RO (UCHL1), CCR7 (G043H7), CD31 (WM59), CD16 (3G8), CD56 (5.1H11), CD127/IL-7Ra (A019D5), CD25 (BC96), CTLA-4 (L3D10), Helios (22F6), CD19 (HIB19), CD27 (M-T271), and IgD (IA6-2), all from Biolegend (CA, USA), and FOXP3 (PCH101) from eBioscience (CA, USA). Cells were analyzed on a BD FACS Aria cell sorter (BD Biosciences, NJ, USA), and analyzed with FlowJo software (Tree Star Inc.; OR, USA).
3
Characterizing CAR T Cell Phenotype
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Mouse anti-human antibodies to CD3 (OKT3), CD4 (OKT4; RPA-T4), CD8 (RPA-T8), CD45RA (HI100), CD27 (M-T271), IFN-gamma (4S.B3), IL-2 (MQ1-17H12), TNF-alpha (MAb11), IL-17A (BL168), CD69 (FN50), PD-1 (EH12.2H7), and TIM-3 (F38-2E2) (Biolegend) were used to evaluate T cell phenotype. Recombinant human GPC3 protein was conjugated to AlexaFluor 647 using NHS ester chemistry and used to detect CAR+ T cells. For all flow cytometry experiments, dead cells were excluded with Fixable Viability Dye eFluor 780 (Fisher Scientific, Cat# 50-169-66). For ex vivo phenotypic studies, rat anti-mouse antibodies to CD45 (30-F11) and CD11b (M1/70) (Biolegend) were used to exclude murine cells from analysis.
To evaluate intracellular cytokine production, CAR T cells were incubated with GPC3+ HepG2 target cells at a 1:1 effector-to-target cell ratio in the presence of brefeldin A (5 μg/mL) and monensin (2 μM) protein transport inhibitors. The co-cultures were incubated for 6 hours at 37°C and 5% CO2 prior to cell staining using the Cytofix/Cytoperm Fixation Kit (BD Biosciences, Cat# 554715/555028).
To evaluate intracellular cytokine production, CAR T cells were incubated with GPC3+ HepG2 target cells at a 1:1 effector-to-target cell ratio in the presence of brefeldin A (5 μg/mL) and monensin (2 μM) protein transport inhibitors. The co-cultures were incubated for 6 hours at 37°C and 5% CO2 prior to cell staining using the Cytofix/Cytoperm Fixation Kit (BD Biosciences, Cat# 554715/555028).
4
Isolation and Sorting of CD8+ T Cell Subsets
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