I am trying to staining algae with PI.
And I used cell cycle protocol.
Fixation with 70% MetOH.
Washing with PBS 2X.
Add RNaseA.
Staining with PI buffered PBS.
But when I spin down pellet of fixed algae to wash, I couldn't see any pellet.
I had thought that it was centrifuge speed problem, so I lowered the centrifugation speed to 300g.
But it was the same.
And maybe, the cell was lysed because of osmosis or their chemical composition after suspended in PBS.
How can i get pellet of algae in washing stage?
Is there any advice for me?
What species are you working with? Some species pellet better than others. It also depends on the algae concentration of course. Sometimes the pellet is almost transparent, have you tried to continue your protocol and see if you have stained cells?
I think you should increase the speed around 2200g, generally, i am using for chlorella
Maybe the following article useful:
Mechanism of Algal Aggregation by Bacillus sp. Strain RP1137
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054200/
I think 70% MeOH can fix and permeabilize algal cells so that PI can get inside the cell. Maybe the key is centrifugation, and 300xg is too low as Ravindra suggested. MeOH can also bleach chlorophylls. Was the supernatant green? If it was, then you might have clear pellet as Carina pointed out.
Carina - I had continued this protocol, but in FACS, the stained cell number was so small that i couldn't make cell cycle peak.
Ravindra - I checked centrifuge step from 1000g to 12000g. But no pellet, no color. So I thought that the contents were all degraded.
Byeong-ryool Jeong - After fixation the supernatant was green and after PBS washing & centrifugation the supernatant was clearly transparent..
Thank you all..
I'll check Jaz & Fikrat's suggestion
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