To begin, take the hBM-MSCs and GFP-HUVECs co-culture and wash once with 200 microliters per well of PBS. Add the BCIP/NBT substrate solution and incubate the cultures at 37 degrees Celsius while periodically monitoring the color development. Once the color develops in non-osteogenic conditions, immediately washed with 200 microliters per well of PBS.
After fixing the samples in 4%paraformaldehyde, acquire color images of the stained samples. To quantify the ALP activity in cell lysates, add 200 microliters per well of 0.25%Trypsin-EDTA to the cultures washed with PBS and incubate at 37 degrees Celsius. Agitate the cultures every 10 minutes by vigorously pipetting up and down to facilitate digestion and monitor the culture morphology with a standard cell culture microscope.
Then transfer the samples to 2-milliliter tubes and add 200 microliters of mesenchymal stem cell basal medium to inhibit the Trypsin. After centrifusing and discarding the supernatant, freeze the pellets at 80 degrees Celsius or proceed directly to the next step. Thaw the cell pellets, add 500 microliters of lysis buffer, and incubate for 30 minutes on ice.
Next centrifuge at 16, 100 G for 10 minutes at four degrees Celsius and keep the samples on ice. Without disturbing any pelleted debris, dispense 50 microliters of the supernatant into each well of a transparent tissue culture 96-well plate in duplicates. Add 50 microliters of the prepared alkaline phosphatase reagent to the wells of the 96-well tissue culture plate.
Shake the plate briefly and incubate it at 37 degrees Celsius for 10 minutes protected from light. Stop the reaction by adding 100 microliters of 1-molar sodium hydroxide using a multi-channel pipette. Using a plate reader, read the optical density at 410 nanometers.
For DNA quantification, thaw the frozen samples, centrifuge them, and place them on ice. Without disturbing any pelleted debris, dispense 50 microliters of the cell lysate supernatant into the wells of a black 96-well plate in duplicates. Add the DNA standards in duplicates.
Next, use a multi-channel pipette to add 50 microliters of the DNA staining agent, then incubate and read the fluorescence intensity according to the manufacturer's instructions. Utilize the standard curve values to determine and apply the conversion of the measured intensity values to the DNA concentrations. Finally, normalize the alkaline phosphatase values by dividing them by the respective DNA concentration of each sample.
Quantification of alkaline phosphatase activity to characterize the osteogenic potential of the cultures showed that the normalized alkaline phosphatase activity varied greatly across conditions with a trend of increasing activity with higher concentrations of BMP-2 and a plateau at 100 nanograms per milliliter. The lowest activity levels were identified for the condition containing 50 nanograms per milliliter FGF-2. More extensive and intense purple staining was observed in the presence of BMP-2 as compared to FGF-2.
