I am isolating AF cells from porcine. Sometimes it happens that cells are live on the basis of live/dead assay by Calcine AM/ethylene Homo-dimer, but do not attached on plates. I have tested the plates and concluded that cells are good for cell attachment. I have treated the tissues fraction with collagenase overnight as reported in papers. Is there any adverse effect on cells after treatment with collagenase for extended periods?
HI, I haven't used AF cells but in my experience primary cells like to grow in high protein media, i.e. high serum concentrations. I tend to start with 20% for the first 24h if cells don't adhere and then reduce it to permissive for the cells concentration. However, it always depends on the cell type, the assays you use etc.
Did you treated the cells directly? Normal way is treating the plates with collagenase and dry the plates. Then seed the cells on the plates.
@ Sven Dittmann,
I have checked the plates and that are good for cell attachment. I use the plates from Nunc Thermo-scientific or BD Bioscience. I have treated the tissue fraction with 0.1% or 0.03% collagenase to separate single cells. As the concentration of collagenase increases, amount of single cell suspension increases. But problem is that cells are not attached on plates where as they are alive.
What type of culture medium are you using?
If using serum-free medium you might need to pre-coat plates with some form of cell attachment factor. I'm not familiar with AF cells. Can you provide more details?
HI, I haven't used AF cells but in my experience primary cells like to grow in high protein media, i.e. high serum concentrations. I tend to start with 20% for the first 24h if cells don't adhere and then reduce it to permissive for the cells concentration. However, it always depends on the cell type, the assays you use etc.
@Demien Tsimbouri,
Respected Sir, I am using DMEM/F12 with 10% decomplemented FBS. I have checked different types of media combination eg DMEM or DMEM/F12 with high /low glucose supplemented with 5, 10 % complemented or decomplemented. All type of combination of media is good for AF cells growth when once I am able to isolate the AF cells.
and recently I have checked that very low percentage of cells, it may be near abut 10-20 cells are attached in 35mm plate after 3-5 days of isolation where as rest of million cells are alive on the basis of LIVE/DEATH assay, with out attaching.
@ Penelope Tsimbouri ,
Dear Sir, Tomorrow I will repeat the experiment and I will follow your advice. Thank you for your suggestion.
Try controlling the collagenase treatment... the longer period / higher concentration you treat, lesser will be the attachment.
High serum may not help in attachment but surely increased the proliferation of attached cells to give you higher density. Try using 20% serum for initial cultures....later you can switch to 10% serum. You may also like to use 20-40 ug/ml EGF for initial cultures.
Further, you probably did not notice the suggestion of Sven correctly.
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