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4 protocols using anti cd62l v450

1

CD4+ Memory T-Cell Phenotyping

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A blood sample of 200 μl was used for CD4+ memory T-cell phenotyping with the following antibodies: anti-CD8-FITC (1/10, clone RPA-T8), anti-PD1-FITC (1/5, clone MIH4), anti-CD122-PE (1/10, clone Mik-B3), anti-CD62L-V450 (1/10, clone DREG-56), anti-CD4-V500 (1/20, clone RPA-T4), anti-CD95-APC (1/10, clone DX2), anti-CD45RA-PE-Cy7 (1/20, clone HI100), anti-CD45RO-PerCPCy5.5 (1/10, clone UCHL1), anti-CCR7-PE-CF594 (1/10, clone 150503), anti-CXCR3-Alexa 700 (1/10, clone 1C6/CXCR3), anti-CD27-APC-H7 (1/10, clone M-T271) (all from BD Biosciences), and anti-CD3-eFluor 650NC (1/10, clone OKT3, eBioscience). After staining, the blood sample was fixed (fix/lyse solution, BD Biosciences) and cells were acquired on a BD LSR Fortessa cytometer (BD Biosciences). Data were analysed with Flow Jo software.
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Multiparametric Analysis of Splenic Lymphocytes

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Splenic lymphocytes were stained for surface markers as follows. For extracellular markers, single cells were stained at 2 × 106 cells per well in a 96-well V bottomed plate. T cells were stained with anti-CD3 Alexa 700 (BD, clone 500A2), anti-CD4 PerCP (BD, clone RM4-5), anti-CD44 FITC (BD, clone IM7), anti-CD62L V450 (BD, clone MEL-14), and anti-FOXP3 APC (BD, clone MF23). B cells were stained with anti-CD19 PEcy7 (eBiosciences, clone 6D5), anti-B220 FITC (eBiosciences, clone RA3-6B2), anti-CD21 APC (BD, clone 7G6), anti-CD23 PE (BD), and anti-CD80 V450 (BD, clone 16-10A1). Fifty microliters of the antibody master mix prepared in MACS buffer (1× PBS, 2 mM EDTA, and 0.5% BSA) was added per well in all staining procedures. Cells were acquired on an LSRII (Becton Dickinson) and analyzed by FlowJo (Tree Star, Ashland).
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3

Detailed Phenotyping of Immune Cells

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Anti-CD14 and anti-CD19 APC-H7 (both at 1:100), anti-CD25 PECy7 (1:50), anti-CD62L V450 (1:50), anti-CD127 PECy7 (1:100), anti-CD161 FITC (1:10), anti-IFNγ V450 (1:200), anti-Ki67 FITC (1:10) were from BD Biosciences. Anti-CD3 ECD (1:100) and anti-CD8β PE (1:50) were from Beckman Coulter. Anti-Vα7.2 FITC (1:10) and PE (1:20), anti-CD45 Alexa Fluor 700 (1:400), anti-CD45RO PECy7 (1:50), anti-CCR9 Alexa Fluor 647 (1:20), anti-Ki67 Brilliant Violet 421 (1:20), anti-CCR7 Brilliant Violet 421 (1:20) were from BioLegend. Anti-CD161 PerCP-Cy5.5 (1:20), anti-IL-22 PerCP-eFluor 710 (1:50) were from eBioscience. Anti-IL-18R PE (1:20), and anti-PLZF APC (1:10) were from R&D systems. Anti-CD4 Qdot 655 (1:400), anti-CD8α Qdot 605 (1:800), anti-CD45 Qdot 705 (1:100), live/dead aqua (1:100) and near infrared fixable (1:400) cell stain were from Invitrogen. Cell surface staining was performed using directly conjugated antibodies and fixed in Cytofix/Cytoperm (BD Biosciences). Intracellular staining was performed using the appropriate mAb’s in Perm/Wash (BD Biosciences). Samples were acquired on an LSRII flow cytometer (BD Biosciences) equipped with 405, 488, 532 and 647 nm lasers. Single-stained polystyrene beads (BD Biosciences) were used for compensation purposes. Software-based compensation was performed using the compensation platform in FlowJo software version 9.5 (Tree Star).
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4

Comprehensive Splenic Lymphocyte Analysis

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Splenic lymphocytes were characterized by flow cytometry. Single cells were stained at 2 ×106 cells per well in a 96 well V bottomed plate with anti-CD3 Alexa 700 (BD, clone 500A2), anti-CD4 PerCPCy5.5 (BD, clone RM4–5), anti-CD19 PECy7 (BD, clone 6D5), anti-CD44 FITC (BD, clone IM7), anti-CD62L V450 (BD, clone MEL-14) and anti-FOXP3 APC (BD, clone MF23). 50μl of the antibody master mix prepared in MACS buffer was added per well in all staining procedures. Cells were acquired on an LSRII (Becton Dickinson) and analysed by FlowJo (Tree Star, Ashland) software.
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