Cells are lost after fixation - (Nov/30/2007 )
I've tested it. With or without Ca & Mg makes no difference. The treatment of fixed cells need to be more gentle during washing
good to know
As far as I know it makes a big difference whether you wash your cells with PBS with or without Ca/Mg...
at least before you fix them, because Ca/Mg are important for cell-cell-contacts for example.
When you stain membrane proteins you really see a difference.
I agree with the others in the point that you should treat them very gentle.
Greetings,
Chakchel
That could be true because also a lot of my colleagues use PBS with Calcium and Magnesium so i will stick to that.
Also, last time when i've added collagen to the wells i've washed them (no cells there) with after 1 hour because i was in a hurry. But after waiting 2+hrs after adding collagen, a larger percentage of cells were fixed so that is a reason for cells not fixed wells.
On another note, does anybody use confocal on Zeiss510 Meta? It's my first confocal ever and i'm starting to learn it. I've found it easy to learn but i need to pracise it and get more experience. What are yourt suggestions?
Also, last time when i've added collagen to the wells i've washed them (no cells there) with after 1 hour because i was in a hurry. But after waiting 2+hrs after adding collagen, a larger percentage of cells were fixed so that is a reason for cells not fixed wells.
On another note, does anybody use confocal on Zeiss510 Meta? It's my first confocal ever and i'm starting to learn it. I've found it easy to learn but i need to pracise it and get more experience. What are yourt suggestions?
Yes, I use Zeiss 510 (Meta sometimes, but mostly just 510), but procedure is quite familiar, but your question is way too broad. Tell me more about what you are doing and what you want to know and I will try to help out (if I can).
glad to hear that your cells are OK now, but it is true that period that we are most likely to lose cells is before fixing. HepG2 tends to grow upwards, so I think that for your case, the seeding time and number of cells when seeding are important, not just confluency; also make sure to passage well to reduce clusters. This could help with taking pictures later. Coating coverslip may help out too. But even so, PBSCM is supposed to help make cells spreadout more and maybe sticking better, For cells that are already spread out well like Hela, it may not be necessary, but HepG2 is different and that could do a little help (maynot much with fixing but with IF later). Regardless what cell line, the first rinsing should be brief and gentle. All washing steps should not be performed by adding buffer directly on the coverslip. Some will even find that pH could help to make cells spread out better.