Acrodisc white blood cell syringe filter

PBLs were isolated from WT mice blood at D8 post-EAE using Acrodisc® white blood cell syringe filter (Pall Life Sciences, Port Washington, NY). Following 4% PFA fixation, PBLs were either left unpermeabilized or permeabilized with 0.2% Triton X-100 for 30 min and incubated overnight at 4 °C with anti-CLN-5-Alexa® 488 antibody (1:150; Life Technologies, Carlsbad, CA). Unlabelled PBLs and PBLs incubated with Alexa® 488-conjugated isotype controls were used for compensation. The intensity of CLN-5 immunostaining in PBL suspensions with or without permeabilization was analyzed on a FACS LSR II (Becton Dickinson, Franklin Lakes, NJ). Data analysis was performed with FlowJo software version 9 (Treestar, Ashland, OR).

Reticulocytes were enriched from heparinized umbilical cord blood with 19% Nycodenz solution (Accurate Chemical & Scientific Co. Carle Place, NY) in high-KCl buffer using gradient centrifugation as described previously (Sorette et al., 1992 (link)). Briefly, fresh cord blood was washed with incomplete RPMI 1640 medium, and leukocytes were removed by Acrodisc^® white blood cell syringe filter (Pall Life Sciences, Port Washington, NY). The packed cells were resuspended in high KCl buffer (pH 7.4) and incubated at 4°C for 3 h while rotating. Five milliliters of the RBC-high KCl buffer mixture was overlaid on 3 ml of 19% Nycodenz solution and centrifuged at 3,000 ×g for 30 min. Reticulocytes were harvested from the interface layer, and the concentration and purity were calculated by observing more than 2,000 RBCs in thin blood smear samples stained with new methylene blue stain solution (Sigma-Aldrich).