Sorted CD8-positive and CD8-negative fractions were stained as described previously40 (link) using the following antibodies: anti-CD8 Pacific Blue (clone RPA-T8, BD), anti-CCR7 FITC (clone REA546, Miltenyi), anti-CD4 ECD (clone T4, Beckman Coulter, Brea, CA), anti-CD45RA PerCP-Cy5.5 (clone HI100, BD), anti-CD20 AF700 (clone 2H7, BioLegend, San Diego, CA), and anti-CD3 APC-H7 (clone SK7, BD). Dead cells were excluded using the Aqua LIVE/DEAD stain kit (Invitrogen AG, Basel, Switzerland). Data were acquired on a LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ). A blinded investigator acquired the data on a LSRII flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar Inc., version 10.7.1). All samples from a given study participant were analyzed on the same day; each set of experiments included pwMS from different baseline, untreated, or previously treated so that potential sources of variation and technical side effects were minimized.
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Flow Cytometric Analysis of T Cell Subsets
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Variable analysis
independent variables
- Sorted CD8-positive and CD8-negative fractions
- Expression of the following markers on the cells: CD8, CCR7, CD4, CD45RA, CD20, CD3
- Staining procedure described previously in reference 40
- Acquisition of data on a LSRII flow cytometer (Becton Dickinson)
- Analysis using FlowJo software (TreeStar Inc., version 10.7.1)
- All samples from a given study participant were analyzed on the same day
- Each set of experiments included pwMS from different baseline, untreated, or previously treated conditions to minimize potential sources of variation and technical side effects
- Positive control: Not explicitly mentioned
- Negative control: Dead cells were excluded using the Aqua LIVE/DEAD stain kit (Invitrogen AG, Basel, Switzerland)
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