Hi everyone , can any one help me in FACS Aria III , I work extensively to analyse cell cycle of cumulus cell but no result
i donot know where is the problem
Sorry, which assay do you use? The PI one or smth else? Can you show the plots you get?
i use this protocol
Reagents
• Propidium Iodide Staining Solution (1mg/ml) [Invitrogen, P1304MP] in PBS.
• RNase A stock solution: 1 µg/ml RNase A [Ambion, USA ] [AM2274]
• 100% ethanol stored in the freezer at -20 °C.
• Wash buffer: PBS.
Protocol
1. Harvest cells and prepare single cell suspension in wash buffer
2. Wash cells twice
3. Centrifuged 12000 rpm for 2min
4. Add 3 ml cold (-20 °C) absolute ethanol to resuspend the pellet
5. Cells stored in the ethanol fixative at -20 °C for several weeks prior to PI staining and flow cytometric analysis).
6. Centrifuged 12000 rpm for 5min
7. Wash cells twice with PBS
8. Add 950µl of PBS
9. Filter the cell suspension using cell strainer (BD Falcon, 40µm Nylon)
10. Add 1 µl of RNase (1µg/ml), incubate for 15 min
11. Add 50 µl of propidium iodide staining solution to cell and mix well. Incubate 3 hr on ice prior to analysis.
Make sure you acquire data in linear, not logarithmic scale. You should get at least 2 peaks; bad staining or instrumentation problems result in one wide peak with long tail
Hi everyone
if anyone know any conferences concerning the oocytes of mammalian species hold this year 2012
Also, 1 peak is not necessarily a deficiency. Many cell types have such a slow cell cycle that most of the cells are in G0/G1 phase, and it may be so much bigger than your G2/M that you think there is only 1 peak. As was stated above, a figure would help troubleshoot this problem more easily.
I'm going to guess that your FSC and PE voltages are way too high, and what you're seeing on the screen here is the amplified debris. If you run this again, bring your FSC voltage down until there is pretty much nothing touching the high end of the axis. Same goes for the PI channel (PE).
In the first set of plots your scatter is too high. Lower the FSC voltage. Also run PI in linear (you can use the PE, PE-TxRed or the PE-Cy5 filter but PE-TxRed is at the optimal emission of PI). As others have said, ise 70% ethanol for fix and final PI concentartion of 20-50ug/ml for mammalian cells.
I would also add that, before doing anything, make sure the instrument is clean and well aligned using fluorescent beads. For example, with FlowCheck 2 um YG beads from Polysciences, the BD machines should present coefficient of variations (CV) of
Thanks all for replying me
i will follow your recommendations hopping to get good results
- Badges
- Science method