Hello,
I am setting up the culture conditions to quantifiy collagen I production in a primary culture of dermal fibroblasts. I am using and ELISA kit to quantify and I get signal in my basal wells but I do not succeed to see an increase in signal with the positive control TGFb1 (10ng/mL), in fact, I see a decrease in signal. The protocol I am following is:
1. plating in 96well plate in media106+LSGS supplement for 24h.
2. Deprivation in media106+LSGS (diluted 1/20) for 24h.
3. Treatment with ascorbic acid 50ug/mL + TGFb1 10ng/mL for 48h ( I have also tested 24h and 72h with same results)
4. Sample collection: I have tried supernatant, pellet (lysed by performing freeze/thaw cycles) and a pool of supernatant and pellet
5. Quantification by ELISA using cusabio kit.
As said, I have signal in my wells but I do not see the increase with TGFbeta1 (it should work according to literature)..
Any recommendation will be appreciated.
Thanks
PC
Hi Patricia,
I may advise you to use more number of fibroblasts (around 2 millions) in T75 flasks, and deprivement by using serum free media for 24 hours. After that you can collect the conditioned media to quantify the required biofactor.
The conditioned media can be collected after 4, 8, 24, 48 and 72 hours, it may a lot of work but stii worthy to give a go.
Feel free for any further qeustions, please
Best wishes
Sam
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA