Flow cytometry staining buffer

BMDCs were treated with Mo-Pr (50, 100, or 200 μg/mL), lipopolysaccharide (LPS) (1 μg/mL) (Biosharp, Beijing Labgic Technology Co., Ltd., Beijing, China), or RPMI 1640 for 48 h. The BMDCs were washed twice and resuspended in a flow cytometry staining buffer (MultiSciences Biotech Co., Ltd., Hangzhou, China). TruStain FcXTM PLUS (anti-mouse CD16/32) antibody (BioLegend, San Diego, CA, USA) was added to the culture of BMDCs at 4 °C for 10 min, followed by three washes with the flow cytometry staining buffer. PE anti-CD11c, PE anti-CD80, PE anti-CD86, APC anti-MHC class II (I-A/I-E), APC anti-OX40L (CD252) (Proteintech Group, Inc., 5500 Pearl Street, Suite 400., Rosemont, IL, USA), and Alexa Fluor^® 674 anti-TIM-4 antibodies (BioLegend, San Diego, CA, USA) were added to the culture of BMDCs at 4 °C for 20 min. After filtration through 200 mesh nylon, the BMDCs were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, Accuri Cytometers Inc., 173 Parkland Plaza, Ann Arbor, MI, USA).

The nHA (purity >97%, CAS 1306-06-5) was purchased from Aladdin Industrial Inc. (Shanghai, China). Cell Counting Kit-8 (CCK-8), Annexin V-FITC/PI apoptosis detection kit, bicinchoninic acid (BCA) protein assay kit, Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum (FBS), penicillin and streptomycin were supplied by Meilunbio (Dalian, China). EasySep™ mouse T cell isolation kit was obtained from StemCell Technologies (Vancouver, CA, United States). Anti-mouse CD3 (Clone 17A2), anti-mouse CD28 (Clone 37.51) were purchased from Peprotech (Westlake Village, CA, United States). Anti-mouse CD4, phycoerythrin (PE, Clone GK1.5), Anti-mouse CD8α, phycoerythrin-cyanine 7 (PE-Cy7, Clone 53-6.7) and flow cytometry staining buffer was purchased from Multi Sciences Biotech, Co., Ltd. (Hangzhou, China). 5-(and-6)-Carboxyfluorescein diacetate, succinimidyl ester (CFSE) was obtained from Abbkine Bioadvisers. Alizarin red S (ARS) staining kit for osteogenesis was purchased from Beyotime (Shanghai, China). Dimethyl sulfoxide (DMSO) and RPMI 1640 culture medium were obtained from Solarbio (Beijing, China). Deionized (DI) water was used in all experiments. All chemicals were used without additional purification.