I am culturing chondrocytes on a 3D PCL based scaffolds and at every 1, 7, 14 and 28 days time period. I would like to quantify DNA or assays the cell proliferation rate. Could I am interested in a protocol that would help me perform this assay without using any kits.
Dear Dr. Jonnala,
The Best strategy is staining the cells with a living dye call CDIL-Traker and than counted manually the cells using confocal microscopy, you can automatically analyze the samples using in my case the Zen (Zeiss edition) 9X9 mm surface layer scanning coupled to Z stacking analysis.
Hopes it help,
Philippe
I have used Alamar Blue assay to measure cell proliferation. The reagent (100 mL) is ~$400 from Life Technologies.
The active ingredient in Alamar Blue is resazurin and you can prepare a solution for the assay without buying the reagent. See reference: J. O'Brien et al., Eur. J. Biochem. 267 5421-6 (2000).
I would agree with Stephen Florczyk that the resazurin assay is great for following cell proliferation in tissues. We have employed the assay for human chondrocytes in PLA constructs in unpublished studies. It has the advantage that it does not destroy the sample so the same construct can be examined over time. This keep costs under control because far less constructs are needed. The method we use is described in the following reference where we used the assay on 2 or 3 days post treatment of endothelial cells (Campbell, L.H., Brockbank, K.G.M. (2012) Culturing with trehalose produces viable endothelial cells after cryopreservation. Cryobiology, doi:10.1016/j.cryobiol.2012.02.006). In the attached reference we used the assay as a viability assay at a single time point after treatment for chondrocytes in porcine cartilage. In follow on studies that have been submitted for publication we used the assay at several time points after treatment of porcine cartilage to determine whether there was any chondrocyte recovery over time under physiological cell culture conditions.
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