Cck 8 solution

100 μL cell suspension per well was prepared in 96-well plates and preincubated for 12 hours in an incubator (37°C, 5% CO₂). After treating with or without CXCR5 siRNA transfection, the cells were incubated with or without CXCL13 at 500 pg/mL for 4, 8, 12, or 24 hours. 10 μL CCK-8 solution (Sangon Biotech, Shanghai, China) and 90 μL medium were added to each well and incubated away from light for 2.5 hours. The optical density of each well was determined by a microplate reader set to 450 nm.

A total of 2*10³ cells in each plate were incubated under the conditions mentioned in the cell culture section in 96-well plates. At 0 h, 24 h, 48 h, 72 h and 96 h, 10 μl CCK-8 solution mixed (Sangon Biotech, China) with 90 μl RPMI 1640 medium was added to each plate and incubated for 2 h at 37 °C. The absorbance at 450 nm was measured (BioTek Epoch, USA).

The cells were seeded into 96-well plates at a concentration of 2000 cells per well in 3 replicate wells after transfection 48 h. At 24, 48, 72, and 96 h, 10 µL of CCK-8 solution (Sangon Biotech) was added to each well. After incubation in a 5% CO₂ atmosphere at 37 ^∘C for 2 h, the mixture was shaken for 1 min on a shaker in the dark. Then, absorbance at 450 nm was measured using a microplate reader. The absorbance value was used as the ordinate and the interval time was used as the abscissa. The CCK8 cell growth curve was drawn using GraphPad Prism 5.0 software.

Isolated ECFCs were cultured to passages 5-7 for cellular assays. Tube formation assays were performed as described previously [56 ]. Briefly, ECFCs or HUVECs were seeded onto 96-well tissue culture plates coated with 30μL Matrigel (BD Biosciences, San Jose, CA, USA) at a cell density of 5x10³ to 2x10⁴ cells/well. Cells were observed every 2 hours by visual microscopy with an inverted microscope (Olympus, Lake Success, NY, USA) at 40X magnification for capillary-like formation. Colony-forming assays were performed as described previously [28 (link)] using limiting dilution assays. ECFCs were seeded in 6-well plates precoated with type 1 rat tail collagen (200 cells/well) with each condition plated in triplicate. On day 14 after initial plating, wells were scored for colony formation by visual inspection with an inverted microscope under 40X magnification. CCK-8 assays were performed for evaluation of cell proliferation. Briefly, cells were seeded in 96-well plates at a density of 10⁴ cells/well and incubated at 37° C for 24 hours. Then 10 μL of CCK-8 solution (Sangon Biotech, Shanghai, China) was added to each well of the plate. After incubation for 1 to 4 hours, the absorbance at 450 nm was measured.

Cells were cultured in 96-well plates and transfected with siRNA. For each time point, 10 μL CCK-8 solution (Sangon Biotech, China) was added to culture medium and incubated for 1 h in the incubator. The absorbance was measured at 450 nm wavelength. Three biological replicates were performed.

HT22 cells were cultured in 24-well plates (2 × 10⁴ cells/well) for 24 h and incubated with CCK-8 solution (10 μL, Sangon, Shanghai, China) at 37 °C for 3 h. Absorbance at 450 nm was subsequently analyzed by using a spectrophotometer (Thermo Scientific, MA, USA).