Hi, I just want to ask regarding flow cytometry method for indirect staining of U87 cells (Glioblastoma) as I'm still quite new in using this method. My objective is to observe the expression of CD133 from U87 cells.
For the control samples, I used Unstained and Isotype control. Right now, I'm facing a problem where my Unstained result is same as Isotype and my stained samples (which is positive samples). The histogram result show that my unstained, isotype and stained have same peak even though I've already tried adjusting the voltage but the result is still the same. Is there any recommended solution for this problem? Also I want to know how do we gate the negative and positive cells population properly by using unstained control as negative? Thank you and looking forward your positive responses.
For primary antibody, I'm using CD133, meanwhile for secondary is Alexa-fluor@488 conjugated. I didn't use any blocking agent. For FACS buffer, I'm using 5% FBS/PBS.
I suggest you change the antibody as there is no difference in staining between blank, iso, and your target staining. The most likely explanation is that you might attempted to use immunostaining antibodies (primary/secondary) with the flow system. While it sometimes works, you need to keep in mind that antibodies to fixed proteins are not the same as antibodies to native proteins; many flow antibodies are generated by native proteins. The easiest thing to test is to fix your cells with 0.5% PFA and redo the staining if it doesn't work change the ab to the flow-specific antibody preferably conjugated with the chromophore so you can reduce the number of washing steps and associated cell loss.
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