Pe mouse anti human cd62l

The following monoclonal antibodies and reagents were used with the indicated specificity and appropriate isotype controls: FITC-conjugated goat anti-human F(ab’)₂ antibody (Jackson, Pennsylvania), PE-conjugated mouse anti-human CD279 (PD-1) antibody (BioLegend, Santiago, CA), FITC mouse anti-human TCRαβ (BD Biosciences, San Jose, CA), FITC mouse anti-human CD25 (BD Biosciences), PE mouse anti-human CD69 (BD Biosciences), FITC mouse anti-human CD45RA (BD Biosciences), and PE mouse anti-human CD62L (BD Biosciences). All cells were washed, resuspended in 100 ​μL PBS containing specific antibodies, and incubated for 30 ​min at 4 ​°C. Data were acquired on a Beckman Coulter Gallios flow cytometer and analysed using FlowJo version 10 (Tree Star, Ashland, OR).

From each subject full blood samples, i.e. PBMCs, were collected in EDTA
tubes. Leukocyte cell surface antigen analysis was performed by flow
cytometry within 1 hour of sampling. Samples were transferred into
fluorescence-activated cell sorting (FACS) tubes and incubated with
antibodies (dilution per antibody 1:20 in sample) against CD3 (BD
Pharmingen APC-H7 mouse anti-human CD3, cat. no. 341110, BD
Biosciences), CD4 (BD Horizon V500 mouse anti-human CD4, cat. no.
560768, BD Biosciences) and CD62L (PE mouse anti-human CD62L, cat. no.
304806, BioLegend) for 30 minutes. Red blood cells were lysed with BD
FACS Lysing Solution (cat. no. 349202, BD Biosciences). Upon
incubation, samples were washed and pellets were resuspended in cell
wash. All steps were performed at room temperature and protected from
light. Readout of CD3, CD4 and CD62L was performed with the BD
FACSCanto II system (BD Biosciences).