Live dead fixable near ir dead cell fluorescent dye

NK cells were isolated from Buffy Coats obtained from healthy donors using the EasySep Human FITC Positive Selection Kit II (Stemcell Technologies) to deplete CD3⁺, CD14⁺, and CD19⁺ cells in the presence of anti-human CD32 (Fc gamma RII) Blocker (Stemcell Technologies). Subsequently, the remaining cells were stained with CD3 FITC (Beckton Dickinson), CD56 PE-Cy7, CD45 Krome Orange (both Beckman Coulter), and LIVE/DEAD Fixable Near-IR Dead Cell fluorescent dye (Invitrogen). Lineage negative and CD56⁺ NK cells were sorted on the FACSaria IIu (Beckton Dickinson), and the purity of NK cells (>95%) was checked during the procedure.

Cryopreserved PB-MNCs, obtained from healthy donors, were thawed and cultured overnight in RPMI-1640 containing 20% fetal bovine serum and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). Next, PB-MNCs were cocultured with UM9 target cells in a 50:1 ratio, in the presence of CD107a-BV421 (Beckton Dickinson), with or without daratumumab 10 μg/mL for 4 or 24 hours. Subsequently, cells were stained with CD45 KO, CD56 PC7, CD138 PE (Beckman Coulter), HLA-DR APC-H7, TIM-3 BV605, CD3 BUV395, and CD16 BUV737 (Beckton Dickinson). LIVE/DEAD Fixable Near-IR Dead Cell fluorescent dye (Invitrogen) was used to determine viability. Degranulation (CD107a positivity) and surface markers were assessed in T cells and NK cell subsets using flow cytometry (LSRFORTESSA instrument, Becton Dickinson).