I having some trouble in the staining part after airlift in ALI cultures when I have used BEGM media from LONZA.
As per the paper the AMEM and EMEM were used for ALI-culture.
I would like to know you have used the complete medium with serum in them when you started ALI or was it done with Serum-free at the point of Air lift.
I have attached the problem that we are facing when use BEGM serum-free to airlift and stain it. So we are not sure if staining is issue or media is an issue. The phase contrast microscope images are suggesting the confluency and TEER value were >400 Ohm /cm2 before we did staining.
Article The Characterization of the Human Nasal Epithelial Cell Line...
Dear Puraskar Ingale,
In primary sinonasal ALI cultures there is no serum used at all. For IHC staining we some times add FCS in the blocking step and our IHC stainings work fine. So I think FCS in the culture should not give you the staining problems, as you might block already unspecific epitopes. The question is whether FCS interferes with your fixation step before adding the antibody. That I don't know. How do you fix your cells? You can test chilled methanol, 4% PFA/PBS or 70% ethanol for fixing step. And are you really sure that your antibody is working?
Best wishes
Puraskar, you won't get differentiation into ciliated cells if you add serum to the media. To me, the images look like pretty normal H & E of undifferentiated cells. BEGM is not typically used for ALI and differentiation, LONZA's BALI is recommended. It has a slightly different formulation than BEGM. Also, patience is really important for differentiation: Three weeks ALI is pretty commonly used for differentiation of airway cells. Best of luck to you - If you switch to BALI, I think you will see success.
Any medium that you usually use, but without serum, added to the top layer should work for your cells. Just add enough on the top layer of your ALI to keep the cells from drying. If you submerge the cells in too much media, they will not differentiate into ciliated cells.
Jennifer, I tried using B-ALI when the airlift. Sorry for the typing the BEGM in the there.
Asma, Did you mean the bottom layer instead of top. I thought in ALI system we remove media from apical chamber and feed from bottom only.
Have anyone done TEER measurements on similar cultures and cells. I am using EVOM2 and it needs really steady hand to get a good read out. Any tips to use EVOM2?
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