I want to calculate Cellular proliferation along with the population doubling time of MSCs cultured onto scaffolds for bone tissue regeneration. Can anyone suggest to me the required procedures and techniques for the aforementioned problem?
Hey Sambit! It depends on the fact, which method is the most convinient one for you (and also available at the lab :D). You can for example directly count the cells (by staining their cell nuclei with PI or DAPI, taking the pictures from at least 10 fields of view via fluorescence microscope and manually or via software like MATLAB count them). The other option is to measure their metabolic activity via metabolic assay (MTT, CCK-8,...).
Are your scaffolds 2D or 3D? You could use an MTS assay (Ex. G3582, Promega) or a PicoGreen assay (Ex. P7589, ThermoFisher) and use image analysis to verify patterns.
Natalie Rubio my scaffolds are 3D scaffolds for bone tissue regeneration.
Markéta Klíčová thankyou for the suggestion. It was really helpful. But can you please tell me how to calculate the population doubling time from MTT or by staining with PI or DAPI.
To calculate population doubling time when using a metabolic assay, you need to first do a calibration curve for the metabolic assay (MTT, MTS, PicoGreen or Almar Blue) to correlate cell number with fluorescence (or optical) signal. You can do this by seeding several, known ammounts of cells and performing the assay. When you then run the metabolic assay on your scaffolds you will be able to correlate the signal you get with a cell number and from the cell numbers you get for each day you can calculate the population doubling time with the formula: DT= t (log2) / (log (Xt) - log (X0))
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