Hey everyone,
I'm interested to do Cell Cycle Analysis of HSCs in diverse organs in different circumstances. I wondered what the best protocol to do it? Is it recommended to check that by BrdU or Ki67 staining?? How Many BrdU injection required to get detectable staining by Flow cytometry??
Thank you in advance
I prefer BrdU for FACS
http://www.bio-protocol.org/e198 (BrdU protocol)
https://www.bdbiosciences.com/documents/BD_FACSVerse_CellCycleAnalysis_AppNote.pdf
Hi:
I think the best way to go is CFSE dilution method (which allows to determine the number of cycles better than BrdU, and even you can use the proliferation/cycling algorithm of FlowJo) but in combination with Ki67 as a dichotomic readout in the same FACS panel (for Ki67 I use the "Foxp3 permeabilization kit/protocol from eBioscience as it provides better nuclei permeabilization than the reagents designed for ICCS). I think this CFSE/Ki67 is way more powerful than BrdU or CFSE alone.
Regards
I could not understand what do you exactly mean by "diverse organs"?? But, if you are going to analyse bone marrow or peripheral blood samples, using PI is simple and easy. Because PI intercalates directly with DNA and RNA, you need to use RNase to prevent false binding. We have used ready to use commercial prep solutions that does not need validation, just the verification, and had very good results. Depending on the system you use, to obtain a commercial evaluation program such as WinCycle or ModFit will help to interpret the results.
I think , propidium iodide is perfect for analysing cell death from different organs. Facs following ModFit is working well enough to get it published.
In terms of BrdU,with Laser Blue Laser
Emit 520 nm
Excite 488 nm.
For details , follow this link -- http://www.ebioscience.com/media/pdf/tds/88/8811-6600.pdf
All suggestion is OK.
It has been completely dependent to your kit which you are using and follow protecol, you can modified it as your experience.
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