Protocol Online logo
Top : New Forum Archives (2009-): : Flow Cytometry

not clear picks - (May/04/2011 )

when I'm doing cell cycle I'm getting the histogram but the lane is not clean but more as saw teeth which make the analysis with modfit problematic
any Idea or suggestions what should I do?
thanks
Michal

-michalcn-

How you did the staining? Did you use RNAase?

-Denis Baev-

Denis Baev on Thu May 5 12:43:35 2011 said:


How you did the staining? Did you use RNAase?


I used the solusion of PI, RNAase and PBS and wait 20 minutes RT, dark

-michalcn-

The staining is Ok. But how did you fix and permeabilize you cells?
"Teeths" on histogram...How many events did you aquire?

-Denis Baev-

Denis Baev on Fri May 6 13:01:43 2011 said:


The staining is Ok. But how did you fix and permeabilize you cells?
"Teeths" on histogram...How many events did you aquire?

-michalcn-

I fixed with 100% ethanol,then centrifuge discard the sup and re-suspend in the PI solution
the count is 10000 events
any Idea?

-michalcn-

Use "frosen at -20" 70% ethanol

Follow this protocol:

1. Harvest, count and pellet cells following standard procedures
2. While vortexing, add 5 ml drop by drop of cold 70% - 80% ethanol into the cells pellet (1-5 x 10e7 cells). Then incubate at -20C for 2 hours
minimum. These fixed cells can be used up to 60 days after fixing (Store at -20C).
-It is a critical step (!)
3. Wash with 2 ml of PBS washing buffer at 1000 rpm for 5 minutes.
4. Aspirate the supernatant.
5. Add 0.5 ml of PBS wash buffer into each tube. add 10 l of PI Staining Solution (Cat. No. 556463)into each tube.
6. Analyze the sample with FACS (immediately)

-Denis Baev-