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Flow cytometry of RFP and CFP expressing yeast cells - (Feb/09/2010 )

I'm thinking about using flow cytometry to measure the expression of CFP and RFP in my yeast cells. Basically, I just need the number of CFP expressing cells vs the number of RFP expressing cells.

1. Will I be able to quantify expression of CFP and RFP directly, without using antibodies?

2. My experiment takes about 35 h, with samples taken each hour. That means I'm unable to do the analysis immediately. Can I just take samples and store them for later analysis? What would I use to store them,cold 70% ethanol?

Thanks in advance


My plan was store the cells in 75% ethanol at -20C. Since I read that cytoplasmic proteins (with CFP and RFP) might leak out of the cells due to ethanol, I decided to fix them first with formaldehyde.

My protocol was this:
1. grow cells in minimal synthetic medium to OD=1
2. spin them down, 3000g, 5 min
3. Resuspend in 1 ml cold PBS
4. Add 1 mL 2% formaldehyde
5. Incubate for 1 h at +4 C
6. Spin the cells down, wash with PBS
7. Spin down again, resuspend in 1 ml PBS
8. Slowly add 3 ml of cold (-20 C) absolute ethanol (for a total concentration of 75% EtOH) while vortexing

At this point white "flakes" appeared in my samples, probably cell aggregates which are impossible to break down. What am I doing wrong?