A total of 2 to 4 × 106 human PBMCs expanded with specific Immuno-STAT or peptide were pretreated with brefeldin A (BFA) and monensin (ThermoFisher), plated in a 24-well plate, and stimulated at a 1:1 ratio with T2 cells (ATCC) that had been loaded with CMV pp65495–503 (NLVPMVATV) or Mart126–35 (ELAGIGILTV) or HIV-1 p17 Gag77–85 (SLYNTVATL; SL9) peptide for 2 h and washed twice. Cells were stimulated for 5 h, washed, stained with Fixed Viability Stain 780 (BD Biosciences), antibodies against CD3 (clone SK7, BioLegend), CD8 (clone SK1, BD Biosciences) and CD107a (clone H4A3, BD Biosciences), and fixed using IC fixation buffer (ThermoFisher). Cells were next washed in permeabilization buffer (eBioscience), stained with antibodies against TNF-α (clone MAb11, BD Biosciences), IFN-γ (clone 4S.B3, BioLegend), and granzyme B (clone GB11, BD Biosciences) for 30 min at room temperature, washed, and analyzed. For representative FACS plots and pairwise marker quantitation, PBMC were stimulated as described above using T2 cells loaded with 100 nM peptide. For peptide titration studies, PBMC were stimulated as described above using T2 cells loaded with 1 × 10–13, 1 × 10–12, 1 × 10–11, 1 × 10–10, 1 × 10–9, 1 × 10–8, 1 × 10–7, 1 × 10–6, or 1 × 10–5 g/ml peptide.
Cd8 clone sk1
Manufactured by BD
Sourced in United States
The CD8 (clone SK1) is a laboratory reagent used for the identification and enumeration of CD8+ T cells in flow cytometry applications. It binds to the CD8 protein expressed on the surface of cytotoxic T cells. This product is intended for research use only and its performance characteristics have not been established for diagnostic or clinical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 clone sp34 2, by BD (5 mentions)
Cd4 clone sk3, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Cd4 clone l200, by BD (3 mentions)
F ab 2 fragment specific antibody, by Jackson ImmunoResearch (2 mentions)
B7 h3 antibody clone 7 517, by BD (2 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Ccr7 clone g043h7, by BioLegend (2 mentions)
Clone fm276, by Miltenyi Biotec (2 mentions)
Cd45ro clone uchl1, by BD (2 mentions)
Phosphate buffered saline (pbs), by Lonza (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Cd4 clone okt4, by BioLegend (2 mentions)
Cd95 clone dx2, by BD (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Cd107a clone h4a3, by BD (1 mentions)
Ifn γ clone 4s b3, by BioLegend (1 mentions)
Tnf α clone mab11, by BD (1 mentions)
T2 cells, by Thermo Fisher Scientific (1 mentions)
13 protocols using cd8 clone sk1
1
PBMC Cytokine Response Assay
2
Multiparameter Flow Cytometry of Immune Cells
3
Multiparameter Flow Cytometry of PBMCs
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The phenotype of PBMCs cell subsets was evaluated by multiparameter flow cytometry. Cells were incubated in the dark for 20 min RT with the following panel of anti-human fluorescent labelled antibodies: CD3 (clone HIT3a, APC conjugated, BioLegend), CD4 (clone SK3, APC conjugated, eBiosciences), CD8 (clone SK1, APC conjugated, BD Biosciences), CD14 (clone 61D3, FITC conjugated, eBiosciences), CD25 (clone BC96, Alexa Fluor 488 conjugated, eBiosciences), CD69 (clone FN50, PE conjugated, eBiosciences), CD45 (clone L48, PerCyP conjugated, BD Biosciences), Foxp3 (clone PCH101, PE conjugatated, eBiosciences), IL-1β (clone CRM56, FITC conjugated, eBiosciences), IL-6 (clone MQ2-13A5, FITC conjugated, eBiosciences), IL-10 (clone JES3-9D7, Alexa Fluor 488 conjugated, eBiosciences), HLA-DR (clone L243, APC.Cy7 conjugated, BD Biosciences), CD33 (clone P67.6, PE conjugated, BD Biosciences), CD11b (clone D12, APC conjugated, BD Biosciences), CD14 (clone MɸP9, Brilliant Violet 421 conjugated, BD Biosciences) and Arginase-1 (ARG-1, FITC-conjugated, R&D Systems). Isotype controls were used for each experiment. After incubation, cells were again washed, resuspended in flow buffer and analyzed using FACSCalibur and FACSCanto II cytometers (BD, Biosciences). At least 5 × 104 events were collected, and the data was analyzed using Summit software (Summit Group Software).
4
Intracellular IL-2 Measurement in T cells
5
Flow Cytometry Analysis of CAR T Cells
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A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10.7 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g. nontransduced T cells or B7-H3-negative cell lines) served as gating controls. CAR detection was performed using F(ab’)2 fragment-specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), and CD45RO (clone UCHL1, BD Biosciences). LM7 and the negative control leukemia cell line BV173 were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). Cells were additionally stained with DAPI (BD Biosciences) to gate for live cells.
6
Flow Cytometric Analysis of CAR T Cells
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A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g., NT T cells) served as gating controls. CAR detection was performed using F(ab′)2 fragment=specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), CD45RO (clone UCHL1, BD Biosciences), and 41BBL (clone 5F4, BioLegend). Tumor cell lines were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). To determine apoptosis, T cells were labeled with annexin V (BD Biosciences) and DAPI (BD Biosciences). The percentages of apoptotic cells were determined by the percent annexin V positive.
7
Quantifying Tumor-Infiltrating T Cells
8
Characterization of Mtb-specific T cells
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Because of the abundance of Mtb antigens already present in granulomas and involved lymph nodes (Gideon et al., 2015 (link)), these samples were not further stimulated with Mtb peptides (Gideon et al., 2015 (link)). Due to low number of cells, uninvolved lung lobes were also not stimulated. All samples were incubated in the presence of Brefeldin A (GolgiPlug, BD Biosciences) at 37°C/5% CO2 for 3.5-4 hours prior to staining. Cells were stained with a viability marker (LIVE/DEAD fixable blue dead cell stain kit, Invitrogen) and surface and intracellular markers. Surface markers include CD3 (clone SP34-2, BD Pharmingen), CD4 (Clone L200, BD Horizon), CD8 (Clone SK1, BD Biosciences) and CD20 (Clone 2H7, eBioscience).
9
Multiparameter Phenotyping of Immune Cells
10
Profiling Immune Landscapes in Glioma Patients
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Fresh tumor tissues were obtained from 20 patients undergoing surgical resection of glioma in accordance with the institutional review board approval at Seoul National University Hospital. Histological diagnosis consisted of 10 glioblastomas, 4 anaplastic oligodendrogliomas, 3 anaplastic astrocytomas, 1 diffuse midline glioma, 1 anaplastic ganglioglioma, and 1 oligodendroglioma. Tumor specimens were collected in RPMI media at room temperature immediately after surgical resection. MACS brain tumor dissociation kit and gentleMACS™ Dissociators (Miltenyi Biotec) were used to dissociate tissue samples within 2 hours following collection, and debris were removed using MACS Debris Removal Solution (Miltenyi Biotec) according to the manufacturer protocol.
Immune cells were stained for surface markers using fluorescence-conjugated monoclonal antibodies including CD45 (clone HI30, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), PD1 (clone EH12.1, BD Biosciences), and TIGIT (clone 741182, R&D Systems). Flow cytometry was performed using FACS LSR Fortessa™ (BD Biosciences), and data was analyzed using FlowJo software (Treestar).
Immune cells were stained for surface markers using fluorescence-conjugated monoclonal antibodies including CD45 (clone HI30, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), PD1 (clone EH12.1, BD Biosciences), and TIGIT (clone 741182, R&D Systems). Flow cytometry was performed using FACS LSR Fortessa™ (BD Biosciences), and data was analyzed using FlowJo software (Treestar).
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