96 well microplate reader

The serum VIP and tissue VIP concentrations were determined with an established available enzyme-linked immunosorbent assay (ELISA) kit (Adlitteram diagnostic laboratories), which measures rat VIP by double-antibody sandwich ELISA. According to the instructions of the manufacturer, the OD 450 was read on a 96-well microplate reader (Tecan Systems, Inc.).

Control or ARL11-depleted macrophages were seeded in 12-well tissue culture dishes (0.15 × 10⁶ cells/well) in DMEM supplemented with 10% heat-inactivated FBS. After treatment of macrophages with LPS (1 μg/ml) or infection with S. typhimurium for the indicated time periods, the quantity of nitrite in the culture medium was measured as an indicator of nitric oxide production using Griess reagent (G4410; Sigma). Briefly, 100 μl of cell culture medium was mixed with 100 μl of Griess reagent. Subsequently, the mixture was incubated at room temperature for 5 min, and the absorbance at 540 nm was measured in a 96-well microplate reader (Tecan). Fresh culture medium was used as a blank in every experiment. The quantity of nitrite was determined from a sodium nitrite standard curve.

MC3T3-LP, MC3T3-BM, and MC3T3-KO cells with a density of 2.0 × 10⁴ cells/well were inoculated in 24-well plates and cultured in differentiation media. The cells were eventually harvested after 3, 5, 7, 9, 14, 18, and 21 days. Alkaline phosphatase (ALP) activities were determined using an ALP kit (Nanjing Jiancheng, China) in accordance with the manufacturer’s instructions. Protein concentrations were measured using a BCA protein assay kit (KeyGEN BioTECH, China). Enzyme activity was quantified via absorbance measurements at 520 nm using a 96-well microplate reader (TECAN, China) and calculated according to protein concentrations. Total protein content was used to normalize ALP activity, and all assays were repeated at least three times.

For analysis of cell growth and biofilm, 1% (v/v) of the cell culture suspension from the pre-culture was inoculated in a 96-well plate and incubated at 37 °C for 48 h without shaking. Cell growth was measured in terms of cell density, determined according to the optical density using a 96-well microplate reader (TECAN, Switzerland). Biofilm formation was analyzed with crystal violet staining using a previously reported method (Au-O’Toole 2011 (link)). Briefly, methanol fixation of biofilm was carried out and 200 μL of 0.2% of crystal violet solution was added to each well and staining was conducted for 5 min for further analysis.

Biofilm biomass was quantified using crystal violet (CV) staining as previously described^{35 (link),36 (link)}. CV is a basic dye, which binds to negatively charged surface molecules and polysaccharides in the extracellular matrix. After biofilm formation (48 h), the biofilm was washed twice with 200 μl PBS and fixed with 100 μl methanol. After 15 min, the supernatants were discarded and the plate was air-dried. The biofilms were then stained with 100 μl 0.5% CV for 20 min. Subsequently, the plates were washed twice with 200 μl PBS to remove excess of CV. Finally, CV was solubilized by adding 150 μl of 33% acetic acid per well. The optical density (OD) at 590 nm was measured using a 96-well microplate reader (Tecan, Männedorf, Switzerland).

RAW264.7 cells were seeded at a density of 3 × 10³ cells per well into 96‐well plates and stimulated with 40 ng/ml RANKL were treated with or without increasing concentrations of UB assess cytotoxicity for 2 and 5 days. Then, the 100 μl MTT solution (5 mg/ml) was added and incubated for 4 h. After aspirating the medium, 100 μl DMSO was added to each well, and the absorbance at 490 nm was measured using a 96‐well microplate reader (Tecan).

LDH activity was detected using a LDH activity assay kit according to the manufacturer's instructions [24 (link)]. Absorbance values were measured at 490 nm with a reference wavelength of 655 nm, using a 96-well microplate reader (Tecan Systems Inc., Oberdiessbach, Switzerland). The experiment was repeated at least three times.