Easysep pe positive selection kit 2

Lymphocytes were taken from the spleen and lymph nodes of C57BL/6, OT-1, and STAT1−/− mice with or without FLT3L treatment. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in 0.5% BSA/PBS (FACS buffer). Cells were stained with PE-conjugated CD44 antibodies in the staining buffer, washed, and resuspended in the staining buffer. To assess the functions of CD44high CD8 T cells, CD44-positive cells were isolated using the EasySep™ PE Positive Selection Kit II (Stem Cell Technologies). The isolation efficiency was confirmed by flow cytometry analysis. For the T cell proliferation assay, lymphocytes (1 × 10⁶ cells/ml) were suspended in phosphate-buffered saline (PBS) and labeled with the CellTrace Violet™ (Thermo Fisher Scientific, Waltham, MA) at a concentration of 10 μM at 37 °C for 20 min. After labeling, an excess of 10% RPMI/FBS was added to the samples to quench the reaction. Following centrifugation and extensive washing, cells were resuspended in 200 ml of 10% RPMI/FBS with T cell stimulant: either α-CD3 (1.25 ug/ml) and α-CD28 (0.25 ug/ml) antibodies for lymphocytes from C57BL/6 mice, or OVA257-264 (SIINFEKL, 10 or 50 ng/ml) peptides for lymphocytes from OT-1 mice for 24 to 48 h. Cells were then harvested for analysis by flow cytometry.

To further separate macrophages from the rest of MNP pool, two different methods were compared: (1) through magnetic bead separation using EasySep™ PE Positive Selection Kit II (Stemcell, Vancouver, BC, Canada); (2) using the capacity of macrophages to attach to a plastic culture plate to separate them from the non-adherent MNP that remain in the supernatant [44 (link),45 (link)].
For the magnetic bead separation, enriched MNP were incubated with the provided selection mix containing anti-mouse IgG1 and mouse IgG1 anti-pig CD163 monoclonal antibody (Bio-Rad, MCA2311GA, IgG1 clone BL1H7) at 1 mg/mL for 15 min at room temperature (RT) following manufacturer’s indications. After separation, macrophages were resuspended in RPMI medium supplemented with 10% inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, referred to as complete RPMI (RPMIc).
For the isolation via attachment, enriched MNP were seeded in a culture plate and incubated for 2 h at 37 °C in a 5% CO₂ in RPMIc. After incubation, the supernatant containing the non-adhered MNP was removed [45 (link)].