Cell proliferation elisa brdu kit

HRPs and HRECs were exposed for 24 h to the four types of EVs isolated from microglial cultures, as previously described. Other microvascular cells were cultured in parallel for 24 h in the presence of the M1 cocktail, with or without the addition of thiamine.
Proliferation was assessed using the Cell Proliferation ELISA BrdU kit (Merck), and apoptosis was assessed using the Cell Death Detection ELISA^PLUS kit (Merck), according to the manufacturer’s instructions.
Cell migration was measured using the colorimetric QCM Chemotaxis Cell Migration Assay (Merck): cells were seeded inside 8 µm pore polycarbonate membranes and exposed to EVs for 24 h. Subsequently, cells still inside the insert were mechanically removed and those that migrated through the membrane were stained. The dye was then extracted, and colorimetric reading was performed spectrophotometrically at 560 nm.
ROS production was assessed by exposing cells in 96 well/plates for 45 min at 37 °C to 25 µmol/L H₂DCFDA (Invitrogen–ThermoFisher, Waltham, MA, USA) in medium without red phenol and FCS. Wells were then washed and fresh medium was added. Fluorescence was measured at 490 nm excitation/520 nm emission at different time points.

MTT assays, to monitor x-fold cell growth over time, were carried out as described previously^{6 (link)}. BrDU assays were conducted using the Cell Proliferation ELISA, BrDU-Kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Crystal violet staining was performed as described previously^{10 (link)}.

IM-HM proliferation was measured as DNA synthesis through the Cell Proliferation ELISA BrdU kit (Merck, Darmstadt, Germany), and apoptosis and necrosis using the Cell Death Detection ELISA^PLUS kit (Merck, Darmstadt, Germany), according to the manufacturer’s instructions.
Cell migration rate was assayed using the colorimetric QCM Chemotaxis Cell Migration Assay (Merck, Darmstadt, Germany), according to the instructions. Briefly, cells were seeded inside 8 µm pore polycarbonate membranes and exposed to M1-inducing conditions. After 24 h, cells that were still inside the insert were removed and those migrated through the membrane stained. The stain was subsequently extracted and transferred to a 96-well ELISA plate for colorimetric reading at 560 nm.
Reactive oxygen species (ROS) production was evaluated by exposing cells in 96-well/plates to 25 µmol/L H2DCFDA (Invitrogen, Waltham, MA, USA) in medium without phenol red and FCS, for 45′ at 37 °C. Wells were then washed and added with fresh medium. Fluorescence was measured at 490 nm excitation/520 nm emission at different time-points.

After 72 h of restimulation as indicated above, a proliferation assay was performed by using the colorimetric Cell Proliferation ELISA BrdU kit (Sigma-Aldrich, St. Louis, MO) according to manufacturer instructions. In brief, 20 µL of 100 µM BrdU was added to the cells, and the cells were incubated for an additional 2 h. After centrifugation for 10 min at 5000×g, the culture medium was removed, and the cells were dried at 60 °C for 1 h and fixed for 1 h with 200 µL of Fix/Denat reagent. Then, an anti-BrdU-POD peroxidase conjugated antibody was added. After 1 h of incubation at room temperature, the cells were washed three times with PBS and incubated with 100 µL of TMB substrate for 15 min, and then the reaction was stopped by adding 50 µL of 2 M H₂SO₄ to each well. The sample absorbance was measured at 450 nm using an ELx808 Ultra microplate reader (Bio-Tek Instruments INC., Winooski, VT) and KCJunior v 1.41.8 software (Bio-Tek Instruments INC., Winooski, VT). The obtained data are presented as the relative cell proliferation to the proliferation of unstimulated cells within a given group. The experiment was performed once in triplicate.