80 % seeded flask, with good cell morphology, couldn't observe cells after centrifugation step in cell passaging. I've followed all the steps with accuracy still can anyone let me know the possible manual error please?
After centrifugation, did you check for pellet formation? I guess you discarded the pellet when throwing your supernatant
So with this kind of problem (unexplained sample loss), think through all the steps and where the sample SHOULD have been, plus where it COULD POSSIBLY have been, after the end of every step. It's actually a little bit easier with cells, because you can see them (versus my good friends DNA and RNA, which usually just look like water.)
The flask was 80% confluent, so obviously the cells grew.
I'm assuming you next trypsinised the cells. The cells SHOULD HAVE been removed from the flask suspended in media, but they MIGHT POSSIBLY have been left behind in the flask. Did you check that the cells actually lifted off the flask and became suspended? If the trypsin is bad, the incubation temperature too low, the incubation time too short, or too little agitation happened at the end of incubation, maybe most of your cells were left behind. If it's possible your flask is still in the bin in the lab, you could check it under the microscope to see if this is the case.
I'm assuming you next transferred the cells to a tube and spun them down. The cells SHOULD HAVE been pelleted, but MIGHT POSSIBLY have been suspended if the centrifuge didn't start, the spin was too short, or the spin speed too low. You could check this by spotting a sample of the supernatant onto a slide and checking for cells in it.
The other possibility is the pellet was just really small and you missed it. The cell pellet from the smallest flask sometimes just looks like a smear on the side of the tube. I'm assuming you checked for a pellet before discarding the supernatant but it's also possible that you aspirated/poured out a loose pellet and lost it that way.
Did you discard supernatant by aspiration or just pour it out directly?
If pellet was observable and you happened to pour the sup. out, the centrifuge speed and times matters in this context. Since I don't have information regarding your method, therefore its hard to offer any suggestion.
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