I have ordered KSFM media kit but the supplier forgot to send the BPE with the package. I need to run an experiment using NP69 as normal panel cells for anticancer drug test. I have FBS laying around, is it advisable to use FBS while waiting for the BPE delivery? The media KSFM has supplement growth factor and human recombinant EGF added.
I'm a little unclear on exactly what you're asking.
Are you asking if it is advisable to culture your cells in FBS in place of BPE until the BPE arrives?
My best guess is, 'Absolutely not.'
1: FBS comes from blood. It provides cells with many things that need to stay alive and it does that rather well... But BPE comes from the pituitary gland and it is mitogenic; it encourages cell division. So, FBS ALLOWS cell division whereas BPE ENCOURAGES cell division. That seems like a large difference to me.
However, I don't know the exact experiments you're attempting...
2: If you are gathering data, then the data you gather using FBS can't be lumped in with the data you gather using BPE... you'd have to start over anyway...
So, perhaps just wait for the BPE and find something else to do in the mean time...
I hope this helps.
Thanks, it was suggested to me by another colleague of mine from other university and it did not work. The NP69 would not attached.
The supplement that comes with the KSFM is required and when it comes to those cells it's advisable to not use FBS. It's the same principle as with primary keratinocytes, you don't add serum to serum-free media. Just wait for the BPE before proceeding.
Adding FBS might also change your cells! I've worked with primary keratinocytes, which might differentiate when adding serum to the medium. I also would never change the ingredients of the culture medium when doing experiments, the data is most likely useless & you can't use it to compare with previous or future data.
Thanks for the replies. I managed to get Defined KSFM with supplement and human recombinant EGF. I have cultured the NP69 according to a protocol provided along with the cell but it does not seem to attached and flatten out. Prior to culturing the cells, I have made sure that I prepared the correct combination of media based on journals and my supervisor recommendations. Is there any other explanation other than the cells or the T25 flask have problems? The cells remained floating, however only minute amount of cells did attached but still rounded.
Did you make sure to coat the flask or dish with Collagen IV and did you add Rock inhibitor in a dilution of 1:1000 to the media just before plating? I find when my primaries don't adhere it's because of those 2 things. Also adding MycoZap PR helps keep the cells free from possible Mycoplasma, fungi, bacterial, and yeast infection.
There was no other modification mentioned in the protocol that was sent to me regarding the Rock inhibitor or collagen IV. Thanks for the suggestion, I will look into it. That could be the solution that I need. This is the NP69 taken at 200x.
Oh yeah those cells are very unhappy. It looks like a couple cells have attached, but are undergoing a lot of stress and not adhering entirely. Collagen will help facilitate the adherence of the cells to the plastic. Basically it gives them something to grab onto. As for the Rock inhibitor it inhibits the function of Rho-associated protein kinases which can wreck havoc on cells on the vital attachment and initial division stages. It will help ensure the survival of cells in their dissociated state and cryopreserved cells. Although this is my experience with primary keratinocytes derived from skin tissues and I'm not sure how much this will effect the NP69 cells in a favorable or unfavorable way.
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