Hi, here I use trypan at 0,02%...

You can take your stock solution and dilute to this concentration. We don't have problems with precipitation and there's no need to filter.

I hope it helps!

Best of Luck,

Jordana.

Hi,

I don't make my own trypan blue but I have a similar problem using an old stock purchased from invitrogen which is now close to finished and at the very bottom of the bottle there are a lot of precipitates/debris/clumps that make it difficult to count cells. Under the scope it doesn't look like salt crystals but rather stringy clumps. I have tried to get around the issue quickly just by pipetting some of the TB into eppendorfs or falcon tubes and allowing it to sit upright until ready for use so that the debris falls to the bottom, and when I need to use it I slowly take from the top of the solution to try and avoid pipetting up precipitates at the bottom. I have also thought maybe a quick centrifugation would work even better but I never bothered to take the extra time since just waiting for precipitates to settle to the bottom works fine for me most of the time. I have also thought of maybe heating but I wouldn't try this since pH and osmolarity will change with temperature and likely end up killing all cells...

I also bought a new stock so I don't have to deal with this again for a long time but I would still like to know if anyone else has experienced this, what is the cause and identity of the clumps, and if it does happen again how I could better eradicate the annoyance.

Thanks,

-Brenda

Hi Dunia

Try to warm slightly it to eliminate precipitate, and then filter.

Question for Jordana, did you check that changing the concentration of TB doesnt change the efectiveness on marking dead cells?

Your method sounds like a good idea O would just like to know if i would have to Adjust for timings or ratio of volumes TB/cell susp?

Thanks!