In my experiment fibroblasts (cell layer) get dettached on treatment with TGF-beta (5, 10, 15 ng/mL) with media color change. TGF-beta from Sigma was reconstituted in citric acid with pH 3.0 and further dilution in buffer containing 0.5% BSA. Fibroblasts were treated with TGF-beta 24 hours after the cells were split to 24 well plate.
Most likely, the pH of the medium and/or deprivation of cell growth factor(s) contributed to detachment. Without knowing the cell number (cells/well), it is harder to speculate which factor played a major role in the detachment.
I agree with Mr. Choubey that we need a more detailed protocol to give proper advice. How long do you incubate your cells with TGF-beta? Are the detached cells still viable or dead? Also, is it really necessary to reconstitute the protein in citric acid? Your buffer has a low pH and could cause a sudden pH shift in your medium effectively killing your cells.
Finally, which TGF-beta from Sigma are you using? My advice is to check every component you are using in your assay for possible contaminations and toxic additives first. Good luck in your experiments!
If you rmedium becomes yellow, you have a pH issue. You may try to equilibrate your TGF solution toward a pH more compatible with cell culture before adding it to the cells. However it depends on your stock solution concentration since you will have to dilute it. Good luck
Thank you all. For cells detachment issue, I preincubated plates with poly d-lysine (150µl/ml). I assumed its excessive cell proliferation for media color change to yellowish so 24 hours later, media with 10% serum was replaced with fresh serum free media along with TGF beta treatment and it worked. Cells were attached and healthy and there was no problem with media color.
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