In this case, I started with 300,000 cells. I used 0.25% trypsin and 750ul media while extracting them. 1ml PBS and 0.1% FCS were used to wash the cells after centrifugation. This is followed by the addition of 70% ethanol ( at -20 degree celsius). Then 20 ul of PI and 5ul of RNASE were added. After 30 minutes of incubation at 37 degree celsius, the cells were analysed using a flow cytometer.
But I am unable to see any cells on the plot. Any suggestions on what could be wrong?
From your description, I am assuming your cells have clumped and hence there is a blockade in the flow cytometry needle which is not able to take up cells. You could try passing MilliQ and Bleach from through your flow cytometer needle to clear our any blockades. You could also cross check with your colleagues if they are facing any issues with detection or if its just you. If its just you then you would be sure that your cells have clumped together. Additionally, you could try the following protocol for further improvements in your experimentation even if my protocol is only slightly different than yours.
PI Staining Protocol:
Prepare 70% ethanol and store it in a freezer (0 deg or -20 deg) before starting the procedure.
1. Harvest cells (300,000 to 500,000 max; do not exceed this number as it generally tends to increase the CV and you'd get broad peaks) by trypsinization.
2. Centrifuge at 1200rpm for 3 minutes.
3. Give 1 PBS wash at 1200rpm for 3 minutes.
4. Fix your cells with 1ml of the 70% ethanol you made above, pipette, and store them at -20 deg C for overnight.
NOTE: Add 70% ethanol drop wise and slowly through the walls of the tube avoiding any clump formation. It can be stored in -20 deg C for atleast a month.
5. Next day, centrifuge at 1200rpm for 3 minutes.
6. Slowly decant the ethanol taking care the cells are not lost during this process as the cells become very loose due to the presence of ethanol.
7. Give 1-2 PBS wash(es) depending on the pellet and the cell loss.
8. Transfer the cells to the FACS tube and add about 300-500ul PI buffer (50ug/ml - PI and 10ug/ul RNAse) to your cells depending on the pellet/cellular density.
9. Incubate at 37 deg C for 15-30 minutes.
10. Proceed with Acquisition.
Hope this will help.
Also, do remember to mix your cells well before acquisition. Perhaps passing them through a 26 or a 30G needle would be the best thing to do so that you are sure that they are not clumping together.
Feel free to ask if you have any more questions.
Good luck~
Trypsin concentration was the same as yours (using 500 ul), but neutralized in 3 ml PBS (lacking Ca2+ and Mg 2+, calling it PBS -/-) containing 4% FBS. Centrifugation at 1500 rpm for 5-7 mins depending on how tight I want the cell pellet. Aspirate supernatant, wash the pellet in 3-5 ml of PBS -/-, and re-centrifuge. Verify that the cell pellet looks good. When I used PI simultaneously with Annexin V, cell pellets were suspended in PBS -/- and stained directly with Annexin V-APC and PI before running FACS the same day.
If the cell samples were to be proceeded later, cells were fixed in 1% PFA for 15 minutes at 4°C, centrifuged, add 2 ml ice cold EtOH, and then tubes stored at -20°C. When starting the staining protocol, I would dilute the EtOH in 10 ml PBS -/- and then centrifuge samples. If you DON't do this, you will not get good cell pellets, because the density of the EtOH prevents the cells to pellet. Then proceed with the staining protocol used. Concentration of PI used was 120 ng/ml and was added to cell suspension just before analysis.
Additional notes: Using FACS tubes that have a cell strainer may also help to reduce clumping, but the most important way to ensure a good cell suspension is to pipet up and down several times before placing samples into FACS tubes.
Did you pass fresh cells to localize them at citometer?
Did you check with a microscope prescence of the cells before passing throwgh cytometer?
Clusters culd be disgregated with vortex at the time of incubation with the colorant (PI or DAPI). Some cells produce extracellular DNA wich should be dissolved with DNasa.
Thanks for the answer Amirali. I have the following questions about the method that you have shared:
1. Do you use 0.25% or 0.05% trypsin to harvest cells?
2. The first time I tried, I did the entire procedure as per my method on the same day. As per my method, I left the cells at 4 degree for an 1hr after adding 70% ethanol. The ethanol used here was stored at -20. But In your method you seem to leave them at -20 overnight and then centrifuge them the next day. What is your opinion on this difference between our methods? Could this contribute to less cell retrieval with my method?
Fixation step is very critical. Fix with 90% chilled ethanol and incubate at -20 overnight. Go ahead as per Amirali's protocol then and pass your cells after staining through a cell strainer and then acquire.
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