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HACAT cells


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18 replies to this topic

#1 cotchy

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Posted 23 April 2009 - 01:00 AM

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?

#2 Bomber

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Posted 23 April 2009 - 01:34 AM

Hi,

the Hacat cells we use here are rather looking good after thawing. The have been stored at -80C for some vial.
Are you sure the DMSO is still fine and has not beeon oxidized or something?

We use only 5% DMSO for freezing (though I doubt that this makes the difference) in regular media.
What we use here is DMEM with lower bicarbonate content so that the media can be used in incubators with 5% CO2 and doesn't become alkaline (pink) in between.
Do you buffer the media with HEPES or anything?

#3 cotchy

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Posted 24 April 2009 - 03:19 AM

Hi,

the Hacat cells we use here are rather looking good after thawing. The have been stored at -80C for some vial.
Are you sure the DMSO is still fine and has not beeon oxidized or something?

We use only 5% DMSO for freezing (though I doubt that this makes the difference) in regular media.
What we use here is DMEM with lower bicarbonate content so that the media can be used in incubators with 5% CO2 and doesn't become alkaline (pink) in between.
Do you buffer the media with HEPES or anything?



Hi Bomber,

I dont get pink media so i pressume i am ok on this point.

I dont use HEPES as i use the 5 % CO2, should i use HEPES also?

Have you tried to use 10 % DMSO for freezing or do you always use 5 %?

Can you tell me your full recipe for the culturing media?

Mine is:

DMEM (no hepes or L-glut)
10 % FCS
0.5 % penn/strep
0.5 % amphotericin
1 % L- Glutamine

Also what is your freeze media?
is it 75 % DMEM
20 % FCS
and 5 % DMSO?

Many thanks for your help.

#4 Bomber

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Posted 24 April 2009 - 10:18 AM

Hi,

the media is DMEM (high glucose) containing L-glutamine but no Hepes.
We use 10% FBS - that is all.
For freezing them we use this media plus 5% DMSO.
Though I haven't tried it with 10% DMSO I doubt that this is a problem for these cells.
Are your sure the DMSO is fresh?

As an alternative you might try to culture/to adapt the cells to regular RPMI media.

Best regards

#5 cotchy

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Posted 28 April 2009 - 12:45 AM

Ok thanks for your advice bomber.

#6 marie

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Posted 29 April 2009 - 06:03 AM

Hy,

Can somebody tell we what the abreviation HACAT stands for. i know that its a keratinocyte cell line but I mean the exact long version of the abreviation.

Thanks!

#7 Chakchel

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Posted 29 April 2009 - 06:55 AM

HaCaT is the abreviation of: Human adult low Calcium Temperature keratinocytes

#8 Neurite

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Posted 03 May 2009 - 10:56 AM

We freeze them in 90% serum, 10% DMSO and directly from the room to the liquid nitrogen.... They are just fine like that when we thaw them.... like 80%... they will fill the dish in 2 days :P

#9 polarka

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Posted 11 January 2011 - 03:52 AM

Hi,
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?

#10 Penguin

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Posted 11 January 2011 - 04:16 AM

Hi,
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?


Hi,

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.

P

#11 polarka

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Posted 11 January 2011 - 08:20 AM


Hi,
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?


Hi,

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.

P


Hi,
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.

#12 Penguin

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Posted 11 January 2011 - 09:15 AM

Hi,
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??

P

Edited by Penguin, 11 January 2011 - 09:15 AM.


#13 polarka

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Posted 11 January 2011 - 11:54 AM

Hi,
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?

#14 Penguin

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Posted 12 January 2011 - 12:24 AM

Hi,

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

P

#15 polarka

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Posted 12 January 2011 - 12:44 AM

Hi,
That is OK, thanks a lot for your comments, they are very important for me.
Good luck with your research.




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