Confocal microscopy with primary cell cultures - (Feb/23/2007 )
I've done quite a bit of confocal microscopy with cell lines, but i wanted to switch to primary cell cultures (splenocytes to eb exact). I cannot find a lot of papers where they do this...has anybody tried it, and are there any major hurdles other than the cost of animals required to do such work on a large scale?
what exactly do you mean confocal microscopy on primary cultures.
Do you mean live imaging of these cells.
Anyway, I think it shouldnt be a problem.
in principle, there is no difference between primary cells and cell lines regarding CLSM; isolation, culturing and transfection are special for each primary cell and are sometimes more difficult than many cell lines
in principle, there is no difference between primary cells and cell lines regarding CLSM; isolation, culturing and transfection are special for each primary cell and are sometimes more difficult than many cell lines
yes, i thought so which is why i wanted to find somebody who has worked w/ splenocytes:)
For anybody who has done such work, how long would splenocytes stay healthy and viable in complete medium , minus any stimulatory signals/ cytokines?
On an other note, I've done confocal microscopy with non adherent cells( in suspensions) and with adherent cells ( on coverslips coated with alician blue). I'm debating which option to go with for splenocytes, which have some adherent and some non adherent cells. I was hoping to find somebody who has done similar work, to get some feedback if possible!
in principle, there is no difference between primary cells and cell lines regarding CLSM; isolation, culturing and transfection are special for each primary cell and are sometimes more difficult than many cell lines
yes, i thought so which is why i wanted to find somebody who has worked w/ splenocytes:)
For anybody who has done such work, how long would splenocytes stay healthy and viable in complete medium , minus any stimulatory signals/ cytokines?
On an other note, I've done confocal microscopy with non adherent cells( in suspensions) and with adherent cells ( on coverslips coated with alician blue). I'm debating which option to go with for splenocytes, which have some adherent and some non adherent cells. I was hoping to find somebody who has done similar work, to get some feedback if possible!
at least for the dcs and macrophages in the spleen they will not be happy in RPMI for more than 6 hrs-after that they start to undergo stress processes and death. As for which to use on cover slips, it really depends on what you are looking for.
[/quote]
at least for the dcs and macrophages in the spleen they will not be happy in RPMI for more than 6 hrs-after that they start to undergo stress processes and death. As for which to use on cover slips, it really depends on what you are looking for.
[/quote]
Thanks for that info! if i want to keep these cell types happy for a longer period of time, say 2-3 days, do you know what could i go with? I;m testing the ability of a particular compound to induce cell death by various methods, i dont want other factors playing in to give me a false positive!
there is serum free media that you can use that should let them live for at least 2 days...we use one from invitrogen.
when doing 5 day mixed lymphocyte reactions with primary splenocyte preps, I use the following media recipe:
500mL DMEM
50mL FBS
5 mL HEPES
5mL L-glutamine
5mL sodium pyruvate
5mL pen/strep
5mL non essential amino acids
500uL Beta-mercapto-ethanol
sterile filter & carefully replace media daily.
I've never tried to see how long I could keep a single prep alive, but probably not more than a couple days..maybe longer if you add a generic stimulator such as ConA.
thanks for the info, both of u!