Cells were first stained extracellularly with specific antibodies against human CD3 (clone HIT3a), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD45RA (clone HI100), CD45RO (clone UCHL1), CD57 (clone NK-1), CD62L (clone DREG56) (BD Bioscience), KLRG-1 (clone 13F12F2, eBioscience) and Tim-3 (clone #344823) (R&D), were fixed and permeabilized with Fixation/Permeabilization solution (eBioscience), and finally were stained intracellularly with anti-tumor necrosis factor (TNF, clone Mab11), anti-IFN-γ (clone B27), anti-granzyme B (clone GB11), anti-Bcl-2 (clone Bcl-2/100), and anti-EZH2 (clone 11/EZH2) (BD Biosciences). Samples were acquired on a flow cytometry analyzer (LSR II; BD Biosciences) and data were analyzed with DIVA software (BD Biosciences). For lentivirus-infected cells, GFP+ cells were sorted with a FACSAria cell sorter (BD Biosciences).
Cd3 clone hit3a
Manufactured by BD
CD3 (clone HIT3a) is a laboratory reagent used in flow cytometry applications. It recognizes the CD3 antigen expressed on the surface of T cells. This product is intended for research use only and its core function is to facilitate the identification and analysis of T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 clone rpa t4, by BD (4 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (2 mentions)
Cd57 clone nk 1, by BD (2 mentions)
Klrg 1 clone 13f12f2, by Thermo Fisher Scientific (2 mentions)
Tim 3 clone 344823, by R&D Systems (2 mentions)
Anti bcl 2 clone bcl 2 100, by BD (2 mentions)
Anti ezh2 clone 11 ezh2, by BD (2 mentions)
Cd8 clone hit8a, by BD (2 mentions)
Cd45ro clone uchl1, by BD (2 mentions)
Cd62l clone dreg56, by BD (2 mentions)
Cd45ra clone hi100, by BD (2 mentions)
Facsaria cell sorter, by BD (2 mentions)
Diva software, by BD (2 mentions)
Cd14 clone m5e2, by BD (2 mentions)
Cd163 clone ghi 61, by BD (2 mentions)
Cd86 clone it2, by BD (2 mentions)
Cd8 hit8a, by BD (2 mentions)
Cd25 clone 2a3, by BD (2 mentions)
Cd127 biotin clone hil 7r m21, by BD (2 mentions)
Cd56 clone b159, by BD (2 mentions)
4 protocols using cd3 clone hit3a
1
Multiparametric Flow Cytometry Analysis
2
Multiparametric Flow Cytometry Analysis
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Cells were first stained extracellularly with specific antibodies against human CD3 (clone HIT3a), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD45RA (clone HI100), CD45RO (clone UCHL1), CD57 (clone NK-1), CD62L (clone DREG56) (BD Bioscience), KLRG-1 (clone 13F12F2, eBioscience) and Tim-3 (clone #344823) (R&D), were fixed and permeabilized with Fixation/Permeabilization solution (eBioscience), and finally were stained intracellularly with anti-tumor necrosis factor (TNF, clone Mab11), anti-IFN-γ (clone B27), anti-granzyme B (clone GB11), anti-Bcl-2 (clone Bcl-2/100), and anti-EZH2 (clone 11/EZH2) (BD Biosciences). Samples were acquired on a flow cytometry analyzer (LSR II; BD Biosciences) and data were analyzed with DIVA software (BD Biosciences). For lentivirus-infected cells, GFP+ cells were sorted with a FACSAria cell sorter (BD Biosciences).
3
Phenotypic Characterization of PBMCs
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Freshly isolated PBMCs were washed once in FACS buffer (PBS supplemented with 3% fetal bovine serum and 0.1% sodium azide) and stained for flow cytometry. Antibodies used; CD14 clone M5E2, HLA-DR clone G46-6, CD86 clone IT2.2, CD163 clone GHI/61, CD3 clone HIT3a, CD4 clone RPA-T4, CD8 HIT8a, CD25 clone 2A3, CD127-biotin clone HIL-7R-M21, CD56 clone B159, all from BD Biosciences. All analyzes were performed on a FACS Calibur and gated on viable PBMCs using 7AAD dead exclusion stain (BD Biosciences, San Jose, CA, USA).
4
Flow Cytometric Analysis of PBMCs and Monocytes
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PBMCs (10 000–50 000 cells) and isolated monocytes (5000–10 000 cells) were immediately stained for flow cytometry for a total of 20 min at 4°C. Due to inadequate sample amount, we were not able to perform all flow cytometric analyses on all patients. Antibodies used; CD14 clone M5E2 (1:10), HLA-DR clone G46-6 (1:50), CD80 clone L307.4 (1:15), CD86 clone IT2.2 (1:15), CD83 clone HB15e (1:15), CD33 clone WM53 (1:10), CD163 clone GHI/61 (1:15), CD16 clone 3G8 (1:20), CD3 clone HIT3a (1:25), CD4 clone RPA-T4 (1:25), CD8 HIT8a (1:25), CD25 clone 2A3 (1:10), CD127-biotin clone HIL-7R-M21 (1:10), CD56 clone B159 (1:10), all from BD Biosciences. Cells were analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA). Analyzes were performed gated on PBMCs (≥2000 events per sample) and using 7AAD dead exclusion stain (BD Biosciences). Blood dendritic cell analyzes were performed using Blood DC enumeration kit according the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). For co-receptor expression, relative mean fluorescence intensity (MFI) was chosen to avoid any variability in antibody batches.
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