Anti cd38 apc

For fluorescence-activated cell sorting or analysis, half million cells were stained with fluorophore conjugated antibodies in a buffer (0.5% bovine serum albumin in phosphate-buffered saline) for 15 min on ice. Antibodies used for analysis and sorting were: APC-anti CD38, FITC-anti CD38, PE-anti CD90, APC-anti-CD11b (BD Pharmingen), and rabbit polyclonal anti-acetylated FOXO1 (Santa Cruz Biotech). Apoptosis was analyzed by annexin-V (BD Pharmingen) staining. Flow cytometry was performed at the City of Hope Flow Cytometry Core.
For Western blot, rabbit monoclonal anti-human SIRT1 (Epitomics), mouse monoclonal anti-Ku70 (Neomarker) were used. To analyze Ku70 acetylation, we pulled down Ku70 from total cell lysate with anti-Ku70 and protein A/G plus-agarose beads (Santa Cruz) followed by acetylation detection with the rabbit anti-acetyl lysine antibody (Cell Signaling).

Analysis of MICA expression on patient-derived PCs was performed by gating the CD38⁺CD138⁺ PC population using the antibodies anti-MICA (clone 159227, R&D Systems, Minneapolis, MN, USA), anti-CD38/APC, and anti-CD138/FITC (both from BD Bioscience, San Jose, CA, USA) as previously reported (16 (link)); samples were acquired using a FACSCanto (BD Biosciences, San Jose, CA, USA) and a FACSCalibur (Becton Dickinson). Analysis of NKG2D and DNAM-1 on NK cells from PBMCs or BM aspirates was performed by gating on the CD45⁺CD138⁻CD3⁻CD56⁺ population using the antibodies anti-CD3/allophycocyanin-H7, anti-CD56/PE, anti-CD138/FITC, anti-CD45/PE-Cy7, anti-NKG2D/APC, or anti-DNAM-1/APC (BD Bioscience). In some experiments, anti-CD16/PerCP mAb was also used (BD Bioscience). In the experiments relative to Figure 4 and Figure S2 in Supplementary Material, all the patients-derived samples were acquired using a FACSCanto (BD Biosciences, San Jose, CA, USA). In the experiments relative to Figure 5, PBMCs derived from healthy donors were incubated with medium alone or serum derived from MGUS or MM patients for 16 h. After harvesting, cells were stained with antibodies from BD Bioscience: anti-CD3/PerCP, anti-CD56/APC, and anti-NKG2D/PE; samples were acquired using a FACSCalibur (Becton Dickinson).
Data analysis was performed using the FlowJo 9.3.2 program (TreeStar, Ashland, OR, USA).

NK primary cells, treated with 4c compound or with DMSO vehicle control for 24 hours, were labeled with anti-CD25 FITC, anti CD38-APC (BD) and analyzed by FACSCalibur cytometer (BD).

Collected 200 μl BM specimens from MM patients and HC, respectively. Subsequently, labeling the specimens with anti‐CD138‐Bv421, anti‐CD38‐APC, anti‐CSF1‐APC, anti‐CD14‐PerCP, anti‐CD68‐FITC, and anti‐CSF1R‐Bv421 antibodies was done (BD Biosciences). Among these, CD68 is an intracellular marker. The marker of intracellular staining was fixed and permeated with IntraSure Kit (BD Biosciences). Eventually, the data of CSF1 and CSF1R were acquired by flow cytometry (FCM) (Beckman CytoFLEX).

B cells were enriched from PBMC from either individual healthy donors or two pooled healthy donors (to increase cell yield) with RosetteSep human B cell enrichment cocktail (StemCell Technologies#15064). The enriched B cells were stained as described above with 5 µl anti-CD19 BUV395, 1 µl anti-CD38 APC, 5 µl anti-CD11c BV421 (All from BD Biosciences) and 10 µl anti-CD27 BV785 (BioLegend) per 1 × 10⁶ cells. Using a FACS Aria Fusion (BD Biosciences), CD19⁺CD11c⁻ B cells were sorted as naive (CD27⁻CD38⁺), memory (CD27⁺CD38⁻/CD38⁺), or plasma cells (CD27^hiCD38^hi). CD19⁺CD11c⁺ B cells were sorted as CD27⁻CD38⁻ B cells. Post sort purity was greater than 90%; B cells from five independent donors were examined. In one of the sorts insufficient cells were obtained from CD11c⁺ B cells and plasma cells to extract data. The RTL of sorted B cell populations were determined using Telomere PNA Kit/FITC for flow cytometry (Dako #K5327), per manufacturer’s instructions.