Cd138 coated magnetic beads

The clinical features of patients with MM were collected from electronic medical records, which included demographics, immunoglobulin subtype, biochemical indexes level, bone lesion status, renal impairment status, and chromosomal abnormalities. The Durie-Salmon stage of patients with MM was assessed according to the criteria of Durie-Salmon stage system for MM.^{19 (link)} The International Staging System (ISS) stage of patients with MM was evaluated based on the criteria of ISS for MM.^{20 (link)} The BM samples of patients with MM were collected before initial treatment, and the BM samples of healthy donors were collected on the enrollment. Bone marrow samples were then processed with gradient density centrifugation to isolate BM mononuclear cells (BMMC). The separated BMMCs were further purified using CD138-coated magnetic beads (Miltenyi Biotec) to obtain plasma cells. Finally, the plasma cells were stored in liquid nitrogen for any further detection.

The demographics, immunoglobulin (Ig) subtypes, biochemical indexes levels, bone lesion status, renal impairment status, and chromosomal abnormalities were collected from electronic medical records. The Durie-Salmon (DS) stage was assessed based on hemoglobin (Hb) level, serum calcium level, bone X-ray result, and low M-component production rate according to the criteria of DS stage system for MM.^{15 (link)} As for the International Staging System (ISS) stage, it was evaluated based on serum β-2 microglobulin (β2-MG) and albumin (ALB) level referring to the criteria of ISS for MM.^{16 (link)} The BM samples of patients with MM were collected before initial treatment, and the BM samples of healthy donors were collected on the enrollment. After collection, the BM samples were processed with gradient density centrifugation to isolate BM mononuclear cells. Then, the plasma cells were purified from BM mononuclear cells with the use of CD138-coated magnetic beads (Miltenyi Biotec; Catalog No. 130-051-301), and the purified plasma cells were stored in liquid nitrogen for further detection.

Human MM cell lines, KMS-28BM, U266, RPMI-8226 and NCI-H929, were purchased from the American Type Culture Collection. All cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (KMS-28BM, RPMI-8226 and NCI-H929) or 15% fetal bovine serum (U266) (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin at 37°C in a humidified incubator containing 5% CO₂. The plasma cells were isolated from bone marrow mononuclear cells, which were derived from the bone marrow samples of patients with MM and healthy bone marrow donors. Briefly, after collection, the bone marrow samples were processed with gradient density centrifugation (865 × g at 37°C for 20 min) for separating the bone marrow mononuclear cells; subsequently, the separated bone marrow mononuclear cells were purified using CD138-coated magnetic beads (Miltenyi Biotec GmbH) to obtain plasma cells (22 (link),23 (link)). The plasma cells were then stored in liquid nitrogen for further analysis. After incubation at 37°C for 24 h, SIRT2 expression in MM cell lines and normal plasma cells (from healthy bone marrow donors that were used as the control group) was determined by RT-qPCR and western blot analysis.

Bone marrow samples were extracted from patients with MM before treatment initiation, and from healthy donors after informed consent was obtained. Density gradient centrifugation (400 × g for 30 min at room temperature) was then immediately performed to separate the mononuclear cells from the samples, and the plasma cells were isolated using CD138-coated magnetic beads (Miltenyi Biotec GmbH) according to the manufacturer's instructions. The levels of plasma cell lnc-TCF7 were detected using reverse transcription-quantitative (RT-q)PCR.

Bone marrow (BM) aspirates were collected from normal controls and patients after obtaining informed consent and were subjected to red blood cell lysis using Versalyse Lysing Solution (cat. no. A09777; Beckman Coulter, Inc.) and mononuclear cell isolation. Mononuclear cells were isolated using a Ficoll gradient (density 1.077 g/ml) with lymphoprep (cat. no. 07801; STEMCELL Technologies, lymphoprep.html" xlink:type="simple">http://www.stemcell.com/products/lymphoprep.html). Subsequently, plasma cells (PCs) were incubated and separated by positive selection using CD138-coated magnetic beads (Miltenyi Biotec, GmbH) according to the manufacturer's protocol. RNA from PCs was extracted to analyze miR-451a levels, and total protein was extracted to assess IL-6R. The remaining BM was used for multiparameter flow cytometry (MFC). The patients were sampled at initial diagnosis and at the indicated time points during the follow-up.

The bone marrow samples of MM patients were obtained before initiation of treatment. The bone marrow samples of HCs were collected when examining their eligibility for bone marrow transplantation. Then, bone marrow mononuclear cells were separated from bone marrow samples by gradient density centrifugation, and CD138‐positive plasma cells were purified from bone marrow mononuclear cells using CD138‐coated magnetic beads (Miltenyi Biotec). After separation, the plasma cells were stored in liquid nitrogen until further detection.