Anti cd56 antibody

Standard 4 hour ⁵¹Cr release assays were performed and percent specific lysis calculated as previously described.^{6 (link)} Unmodified K562 cells were included as a positive control target and the myeloma cell line OPM2 was included when primary myeloma cells were unavailable (American Type Culture Collection, Manassas, VA). Effector cells were freshly cultured (not cryopreserved) expanded NK cells or NK cells enriched from PB samples or BM using microbeads coated with anti-CD56 antibody (Miltenyi Biotec). Research grade expanded NK cells were produced as previously described.^{6 (link)} CD138-positive plasma cells were purified from BM aspirates via positive selection with anti-CD138 coated microbeads (Miltenyi Biotec). After selection, purity of >90% was confirmed by flow cytometry. For blocking studies, isotype control antibody or anti-KIR antibody was added to effector cells. Control antibody, elotuzumab, or anti-HLA Class I antibody was added to ⁵¹Cr-labeled targets. Antibodies were left in the wells for the duration of the assay, at a final concentration of 10μg/mL.

Primary and immortalized control, FSHD1 and FSHD2 skeletal myoblast cells were grown in high glucose DMEM (Gibco) supplemented with 20% FBS (Omega Scientific, Inc.), 1% Pen-Strep (Gibco), and 2% Ultrasor G (Crescent Chemical Co.). Primary control and FSHD2 (4qA161, SMCHD1 mutation: g.2697999_2698003del) myoblasts (Jiang et al., 2020 (link)) as well as FSHD1 (4qA161, 2 D4Z4 units) myoblasts were immortalized using hTERT with p16INK4a-resistant R24C mutant CDK4 (mtCDK4) and Cyclin D1 as previously described (Shiomi et al., 2011 (link)). After immortalization, CD56-positive cells were selected by magnetic-activated cell sorting conjugated with anti-CD56 antibody (130–050-401, MiltenyiBiotec). Single cell clones were isolated by FACS sorting into 96 well plates. Control, FSHD1 and FSHD2 clones were chosen for the experiments based on normal doubling time and high differentiation index. Myoblast differentiation was induced as previously described (Zeng et al., 2016 ). Briefly, cells were plated at a seeding density of ~2.5 × 10⁵ cells/ml in 0.5 ml of growth medium in each well of a 24-well dish. Approximately 12–16 hr later differentiation was induced using high glucose DMEM medium supplemented with 2% FBS and ITS supplement (insulin 0.1%, 0.000067% sodium selenite, 0.055% transferrin, 51300044 Invitrogen). Fresh differentiation medium was changed every day.