Hello Abdul Mannan

My suggestion is you need to generate something called the "The antibiotic kill curve". Using puromycin concentration as per literature is not the right way I feel since there are a lot of other factors regulating your experiment.

The following link will help you understand better:

https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/antibiotic-kill-curve

Best!

I suspect the problem is one of two things:

The first: the concentration of the puromycin that you are using, look for the appropriate concentration in the literature, especially with the cells that you use. If this is not possible, use more dilute concentrations of puromycin and gradually increase it and note its effect on the cells with transfected cells and other cells.

The second: (pGFP-C-shLenti Vector). There may be an error in the sequence of puromycin resistance gene contained in the vector.

Try using a vector containing the puromycin resistance gene from another source if possible, or try transfection of the vector in another cell type and observe the effect of the puromycin afterwards.

Note: After the success of the transfection and the appearance of GFP, do not use the puromycin on the cells directly. Rather, culture and transfer them to more than one flask, store some of them at -80 for use again when needed. Designate a plate or 96 well plate to treat the cells with puromycin and try different concentrations.

Hi Abdul Mannan , I can only second what they said. Set up a kill curve to measure its effect on your cells. For instance, I use 0.25-0.5ug/mL puromycin. In return, may I ask which method you used for transducing MV4-11 cells? Thanks!

Thanks everyone.

Viola Erdelyi I did spinoculation at 2000 RPM for 2hours

Abdul Mannan , Thanks for the info!