123count ebeads counting beads

ASC_NS-CM was supplemented with 100 ng/ml KC (R&D Systems) as positive control, and with 25 ng/ml monocyte chemotactic protein 1 (MCP-1) or 1 ng/ml IL-1β as negative controls. CMs were added to the lower compartment of 3 μm transwell inserts (Costar). 500,000 freshly isolated primary BM-PMNs in ASC_NS-CM were placed in the top compartment of the transwell inserts and incubated for 2 h. Migrated cells in the lower compartment were collected and quantified with flow cytometry after addition of 123count eBeads™ Counting Beads (Thermo Fisher Scientific).

Cells were suspended with FACS buffer (1 mM EDTA, 0.01% sodium azide, 0.1% bovine serum albumin (BSA), 0.02 M phosphate, 0.15 M NaCl, pH 7.2) and then stained for 20 min with anti-FcR/anti-CD16+CD32/Fc Block (eBioscience) and fluorophore-conjugated antibodies: anti-CD45-PerCP Cy5.5 (BioLegend), anti-Ly6G-AF700 (Thermo Fisher Scientific), anti-CD19-APC (BioLegend), anti-CD11b-PE (BioLegend), anti-NK1.1-PE Cy7 (BioLegend), anti-TCR-beta-Pac Blue (BioLegend), anti-CD4-FITC (BioLegend), and anti-CD8-APC/Cy7 (BioLegend). After staining, cells were washed and then fixed with 1% PFA for 30 min. After fixation, cells were washed three times with 200-μl FACS buffer, and then 50 μl of 123count eBeads Counting beads (Thermo Fisher Scientific) were added to allow for quantification of total number of immune cell subtypes following manufacturer’s instructions. Data were acquired on a LSRII instrument (BD Biosciences). Analysis was performed using FlowJo software, version 10.0.8.

The target cells were labeled with 0.75 µM of CellTracker™ CMFDA (5-chloromethylfluorescein diacetate) Dye (Thermo Fisher Scientific). Co-culture assays were carried out by incubating anti-BCMA-CAR T cells and un-transduced T cells (UTD T) with CMFDA-labeled target cells for 12 h at effector to target (E:T) ratios of 1:1, 5:1, and 10:1. Following co-culturing, counting beads (123count™ eBeads Counting Beads; Thermo Fisher Scientific) were added to each sample to enable quantification of the absolute number of target cells by flow cytometry analysis, using the manufacturer's instructions. The percentage of tumor cell cytotoxicity was determined using the formula: [1 − (target cells in each condition/target cells alone at the indicated time)] × 100. To assess the proliferation activity, anti-BCMA-CAR T cells and UTD T cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) and co-cultured with target cells at E:T ratio of 5:1 in the absence of exogenous cytokines. After 5 days of co-cultivation, CFSE dilution due to cell proliferation was evaluated by gating the lymphocyte population using flow cytometry.