HACAT cells - very low cell viability after thawing cells (Apr/23/2009 )
Penguin on Tue Jan 11 12:16:24 2011 said:
polarka on Tue Jan 11 11:52:17 2011 said:
I would like to seed and grow HaCaT cells to form monolayer. How long time does it take to them to be confluent? Does anybody have experiences with this issue?
It all depends on how many you seed to start with, of course the more cells you put in the faster they'll get to confluency, but also HaCaT cells do not like being at low density. They take longer to double if you seed at 10% then if you seed at 50%. I usually seed 2 X 105 in a 9.5 cm2 plate and by the next day they are at 60% confluent.
thanks a lot for your reply .
Do you also have some experiences with staining the cells when they are in Transwell? I would like to stain iron nanoparticles in the cells. I want to follow:
1) wash with PBS or NaCl
2) 15-30 min 1:1 1N HCl and 2,5% K4((Fe(CN)6)) in H2O
3) wash with distilled water
4) 5 min counterstain with nuclear fast red
5) dehydrate with graded alcohol
6) mount with histological mount
7) visualize by optical microscope
I know these steps, but how really to proceed them-I am not sure. I was thinking about points 1),3),5) just to rinse the membrane with the cells with maybe 5 mL of PBS or NaCl, dist.water, graded alcohol for maybe 2 times from each side of the membrane. But maybe it is too harsh for the cells...Maybe it is better to dip the inserts to these liquids or to pipete them to both chambers what of course will take much more time.
For points 2) and 4)is probably the best to place inserts back to Transwell and fill both chambers with stains. Thus cells will be covered instantly during the exposure.
After all these steps I will cut the membranes with scalpel and somehow place the membranes on the slides and with a few drops of Pertex I will mount them and covered by cover slides.
I will really appreciate if you please could write me what is your opinion about this.
Sorry I have never worked with HaCaT in transwell but your protocol looks good to me. I don't think you need to worry about being too harsh with the cells as they usually stick very hard to substrate - do they stick well to your membrane? Can you do your washing steps in the transwells so that you don't break the membrane i.e. fill the wells with PBS/water/alcohol then aspirate the liquid off??
I did not try to stain the cells yet, therefore I am not sure if I will not be too harsh to them. But I will try and will see if I need to be more gentle-if yes, then I will fill the wells and aspirate the liquid off. These days I am trying to grow the cells. Later, when I am sure how to take care about them I will expose them to nanoparticles and stain them.
You are probably working mostly with HaCaT cells, as I can see your answers here. Are you also publishing articles related to the HaCaT cells?
I really need to study a lot about the HaCaT cells, how to be sure that they form monolayer and not multilayers, how to maintain the best growing conditions for them, about transport of compounds through the cells...Do you have some ideas abou good articles related to the HaCaT cells?
No sorry, I only ever used them as a control cell line to test the off-target effects of the compounds I made, so I don't really know anything specifically about HaCaT characteristics
That is OK, thanks a lot for your comments, they are very important for me.
Good luck with your research.
do you have experience with TEER measurements on HaCaT cells? I measured it, but the values were the same for blanks as well (transwell without HaCaT cells had the same TEER value as Transwell with HaCaT cells). Do you know what could be the reason?
I was thinking that HaCaT cells were not grown as monolayer and therefore TEER could not be measured - because I think that TEER is possible to measure only on monolayer. Do you have explanation?
cotchy on Thu Apr 23 09:00:19 2009 said:
Hi all, a colleauge of mine has recently started to work with HACAT cells.
He bought them in a while ago from CLS (cell line company) at a low passage number and passaged them a few times and then froze them down in the - 80C (70 % DMEM, 20 % serum, 10 % DMSO) and then after a few days places them in liquid nitrogen
When he brings them up from the liquid nitrogen (in the proper recommended media - DMEM w/ 10 % serum ) for use the viabilty of the cells is always very low, around 2 - 5 % and thus it takes 2 to 3 weeks to get a T 25 confluent.
Does anybody have any suggestions as to what is causing the low viability of the cells or has anybody had the same problems and managed to overcome them?
A US company sells the same cells, here is the link http://addexbio.com/productdetail?pid=117. Check that out to see if they also come up with culture condition for this cell line.
For development of TEER, you have to incubate the HaCaT cells with medium that contains more calcium than the usual HaCaT medium to induce further differentiation. After a few days the tight junction network should be well-established.
Just started to grow the HaCaT cells since we bought it a month ago from addexbio http://www.addexbio.com/productdetail?pid=117 and the cells grows wonderfully. Highly recommend their cells. Customer service is great too. Planning to buy another cell line from them.