I did primary culture with caudal fin of fresh water fish according primary fish culture protocol with L-15 media and 15% FBS but the cells did not attach,could you please help me if you have any experience?
Hi Maryam.
To overcome the attachment problem I would suggest two things
1. Reduce the FBS concentration to 10% and
2. Culture the cells at 28 °C in humidified CO2 incubator.
I am summarizing the culture protocol and you can find detailed in the attached paper.
1. Dissect the caudal fin and mince to approximately 2 mm2 in 10cm diameter Petri dishes.
2. Transfer the tissue fragments separately into 50 mL-beakers containing 12 mL of calcium and magnesium free phosphate-buffered saline (PBS), 0.25% trypsin and 0.2% ethylenediaminetetraacetic acid (EDTA) for tissue dissociation.
3. Dissociate the tissues by stirring at 600 rpm and 25 °C for 15 min (caudal fin)
4. Transfer the resulting tissue suspensions into 15 mL centrifuge tubes containing 3 mL foetal bovine serum (FBS) for trypsin neutralization, and centrifuged at 200 g at 25 °C for 10 min.
5. Resuspend cell pellets, including undissociated tissue fragments, in Leibovitz-15 medium (L-15; Gibco) supplemented with 10% FBS, 100 IU mL−1 of penicillin and 100 μg mL−1 streptomycin.
4.Seed the resuspended cells into a 25 cm2 tissue culture flask and incubate at 28 °C in humidified CO2 incubator. One-third of the medium should be replaced with fresh complete L-15 every 14 days until the attached cells reach 90% confluence.
Good luck-
Hello Mandal,
I am agree with the above answer. you have to decrease the concentration of FBS to 10% or a little low then that. it will be helpful for you.
good luck.
thank you so much for your complete answer but i should do all those steps at RT or at 4 C?
Hi Maryam,
Thanks for the message and i am glad that my ideas seem logical to you.
I have gone through the link for EMEM (ATCC) and since you do not have access to a CO2 incubator you probably need a modified EMEM supplemented with 20 mM HEPES to control the pH. CO2 is required to buffer the media containing bicarb. If you reduce the bicarb and increase the HEPES that will help you to culture cells without a CO2 incubator.
You can have commercially available modified EMEM at
http://www4.mpbio.com/ecom/docs/proddata.nsf/(webtds2)/12334
or anywhere else. Just try to match the composition.
There is a recent (funny) thread in RG for creating 5% CO2 atmosphere inside an incubator with no supply of external CO2. You can go through it where I myself and others have been contributed. That will also help you finding an answer.
https://www.researchgate.net/post/Any_tips_for_how_can_I_make_a_5_CO2_atmosphere_inside_an_incubator_with_no_CO2?ev=tp_feed_post_xview
Best-
I have developed the Atlantic salmon epithelial cell line and skin explant in L-15 medium It is recommended
Arriving lately to the discussion but you can check a fairly simple protocol here Article Cell migration analysis: A low-cost laboratory experiment fo...
Best,
D
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