I have samples of frozen tissue supernatant that are being analyzed for cytokine production. However, the cells have been treated with trypsin. Will this affect the concentration of cytokines once the samples have been thawed for multiplex ELISA?
Normally the cells are washed with trypsin to detach them, then trypsin is removed or inactivated and fresh media is added. So there shouldnt be significant amounts of functional trypsin in the supernatant of trypsin-teated cells.
But you say tissue supernatant. Does this mean that you are not talking about cultured cells? How was the tissue tratment done? What do you call tissue supernatant?
Dear Alison Ricafrente
Hello
Pls. find the attached files
http://www.ncbi.nlm.nih.gov/pubmed/10881931
http://www.jimmunol.org/content/175/4/2635.short
http://europepmc.org/abstract/med/11600250
https://www.researchgate.net/publication/11750626_Effect_of_trypsin-chymotrypsin_Chymoral_Forte_DS_preparation_on_the_modulation_of_cytokine_levels_in_burn_patients
http://www.ncbi.nlm.nih.gov/pubmed/18047634
Regards
Prof. Houda Kawas
Article Effect of trypsin-chymotrypsin (Chymoral Forte DS) preparati...
Yes, what Gertrudis says. We're confused what "tissue supernatant" might be, how it's been treated, and if there's really trypsin there.
If you're sure that there is trypsin there, in my opinion you should not trust these samples. Trypsin chops too frequently. It will affect many cytokines. ELISA cannot see them if the 2 epitopes are severed. These samples might be analyzed by mass spec, which naturally includes a trypsin digestion step.
I am using fresh rat spinal cord samples that have been homogenised in 300ul of HBSS. To free the immune cells I added 50ul of a made up solution of trypsin and collagenase
Then your sample is the supernatant after homogenization, not the culture supernatant after growing any cell from your sample. Is that right? If so, trypsin could have degraded any protein in the samples, and these samples arein principle not useful to detect cytokines.
Both trypsin and collagenase must be removed from the medium before analyzing cytokines by ELISA. Before thawing the samples, you must centrifuging to collect the immune cells in the pellet and wash twice by adding 1 mL of fresh HBSS and centrifuging. Finally, the last pellet must be resuspended in HBSS and stored at -80ºC until use.
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