Paraformaldehyde p0099

Cell viability was quantified using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded into 96-well plates at a density of 3x10³ cells/well and were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. Subsequently, 10 μl CCK-8 reagent was added to each well. The absorbance was measured at 450 nm after 2 h. The CCK-8 assay was performed every 24 hours for 5 consecutive days.
For the EdU cell proliferation assay, cells were plated into 96-well plates at a density of 4.0×10³ cells/well and cultured at 37 °C for 48 h. Then cells were incubated with 50 μM EdU solution (Guangzhou RiboBio Co., Ltd.) for 2 h. The wells were washed with PBS, and the cells were fixed with 4% paraformaldehyde (P0099; Beyotime Institute of Biotechnology) at room temperature for 30 min and stained according to the manufacturer's instructions. A fluorescence microscope (Leica Microsystems GmbH) was used to image cells. The number of EdU-positive cells was quantified as follows: (EdU-positive cells/Hoechst-stained cells) ×100%. All experiments were performed in triplicate.

The proliferative ability of cells was evaluated using colony formation assay. Briefly, 1 × 10³ cells were collected and added into each well of 6-well plates. Then, the cells were cultured for 14 days, and the medium was refreshed every 2 days. Next, the culture medium was removed and the cells were fixed by 4% paraformaldehyde (P0099, Beyotime, Shanghai, China)for 10 min. After being washed twice with PBS, the cells were further stained by 1× crystal violet staining solution (KGA229, KeyGEN BioTECH) for 15 min. Finally, the stained cells were rinsed three times by PBS and further photographed by a C-LUX camera (Leica).

Eighteen MRL/lpr mice (20–30 g, female, 6–8 weeks old, purchased from SPF Biotechnology Co., Ltd.) were divided into groups that received a single high‐dose tail vein injection of miR‐99a‐3p agomir, antagomir or NC at an injection volume of 200 µl; miR‐99a‐3p agomir and antagomir injection doses were 20 nmol and 200 nmol, respectively.¹⁵, ¹⁶ Six C57BL/6J (C57) mice (20–25 g, female, 6–8 weeks old) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.
Venous blood was collected from the eye, and B lymphocytes were separated using the immunomagnetic bead method, followed by Western blotting and RT‐qPCR analyses. The plasma was retained for ELISA. Mice were euthanized via cervical dislocation, and their kidneys were removed and placed in 4% paraformaldehyde (P0099, Beyotime, China) for fixation. The experimental protocol was approved by the Animal Research Committee of Kunming Medical University (kmmu2021724).