Phosphate buffered saline (pbs)

Under sterile conditions, 5 ml bone marrow from patients with multiple myeloma and control group was extracted and added with heparin (1:10). The fresh bone marrow specimens were carefully added on the lymphocyte separation medium. Then it was centrifuged for 10 min, at 1,500 × g. The mononuclear cells in the middle layer were collected. After centrifugation for 10 min at 700 × g, the supernatant was absorbed and then precipitated by 1X PBS (Beckman Coulter, Brea, CA, USA) and washed 3 times to obtain cells.

Fresh feces were aliquoted in a CO₂-rich/O₂-low atmosphere and stored at −80°C. Then microbiota were isolated by gradient purification under anaerobic conditions, as previously described (Juste et al., 2014 (link)). In brief, a density gradient of Nycodenz solution was prepared. Then, thawed stool was diluted in 1X-PBS (Eurobio), 60% Nycodenz, and 0.03% sodium deoxycholate and loaded on the gradient. After ultracentrifugation (45 min, 14,567 ×g, 4°C; Beckman Coulter ultracentrifuge), fecal bacteria were extracted and washed three times in 1X-PBS and 0.03% sodium deoxycholate and centrifuged. The final bacterial pellet was diluted in 1X-PBS-10% glycerol, immediately frozen in liquid nitrogen, and stored at −80°C.

We collected 4 mL of fasting peripheral venous blood from all HCC patients before GSM–TACE, 10 days after surgery, and 30 days after surgery using heparin anticoagulation tubes. We then added 100 μL whole blood to a dry blank tube, lysed it with 500 μL OptiLyse C Lysing Solution (Beckman Coulter Diagnostics, Brea, California, US), vortexed the solution, and incubated the lysed blood at room temperature for 15 min to assure lysis was complete. Then we added 2 mL phosphate-buffered saline (PBS), and we vortexed and centrifuged the mixture at 300 g for 5 min. After centrifugation, we aspirated the supernatant, resuspended the cell pellet in 500 μL PBS, added 5 μL each of CD14, CD11b, and HLA-DR antibodies, and mixed the solution low speed for 5 s. Finally, we analyzed the samples on a flow cytometer.
2.2.2 Antibodies and laboratory equipment: To determine MDSC frequency, we performed multicolor fluorescence-activated cell sorting analysis using Beckman Coulter CytExpert software version 1.1. We also used a DxFLEX Flow Cytometer (Beckman Coulter), a Labofuge 400R Centrifuge (Thermo Fisher Scientific, Waltham, Massachusetts, US), a LP Vortex Mixer(Thermo Fisher Waltham, Massachusetts, US), and the following anti-human monoclonal antibodies: CD11b-APC-Alexa Fluor 750, CD14-PC7, anti–HLA-DR-ECD, OptiLyse C solution, and PBS (all Beckman Coulter).