I am having difficulty sorting fixed human sperm on a FACSAria II. After sorting, my cells become very fragile, when I centrifuge at as little as 100g they fall apart and disappear from my solution. Even when I leave them in the fridge for a few days in PBS, even though they are fixed, they fall apart. I am using a 70um nozzle, 34.50 psi sheath pressure and flow rate ~ 2000 events/sec. I have copied these parameters from two papers that have sorted fixed sperm on the same machine. I have tried different fixations; 2% and 4% paraformaldehyde and 100% methanol all with the same result. For this experiment I need to keep my cells intact and be able to centrifuge them.
Any advice is much appreciated!
Thank you
Thw speed of the sort shouldn't be an issue since sperm is very small. However, one way to go would be to try 100 um nozzle and lower sorting pressure, just in case. What are you sorting your sperm into? Do you coat recipient tubes with protein to avoid charged droplets from charging the plastic? This could be an answer to you rpoblem. I usually put media with 10% FSC for one hour on orbital shaker so that whole tube is coated with protein.
I coated the tubes in PBS with BSA and it worked! The cells are staying intact after centrifugation. Thanks so much for your help :)
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