I want to determine TNF-alpha levels in my cell culture. The protocol mentions that i should use Cell Culture Supernatant for this test. There is no mention for the use of any lysis buffer. So does this term refer to medium in which my cells were grown and treated in? or should i prepare a cell lysate?
I carried out this assay on the medium, very low to non TNF-alpha was detected.
Hey Jhoti,
i did a lot of ELISAs and Multiplex Assays detecting different cytokines (of course TNFa too). I used serum-free medium or serum-reduced medium to cultivate my cells and for the collection of the supernatant. While collecting it i cooled the 1,5 ml tubes on ice, pipeeted the supernatant in and centrifuged them at 110000g 15 min and 4 degrees. Then i carefully transferred a bit more than half of the supernatant into new cooled-down tubes and put them in liquid nitrogen before store them at -80 prior to use. I was always able to detect TNFa in the supernatant.
Short answer is "Yes'
You will want to centrifuge your cells in the medium that you grew them in and then remove the supernat to a new tube and use that for your ELISA or whatever test you are doing.
Remeber the time you culture them in the medium (take into account medium changes), the amount of medium you are growing them in, and the confluency of the cells is all going to affect the TNF-alph levels in your cell culture supernatant.
The cell culture supernatant is the media in which the cells were growing. You may want to centrifugate it just to eliminate any debris or floating cells and take just the supernatant without any cell. TNF-alpha should have been secreted to the media.
Hi,
You can use the medium after removing your cells from the culture plate ( by briefly spinning your plate, then collect supernatant). You can try using different stimulation conditions, and different time points for collecting the cell culture supernatant to get optimal concentration of TNFa secretion.
Good Luck!
The explanation by Monica Delgado is the best: the cell culture supernatant is intended to be free of any (floating) cells possibly detached from the monolayer culture you collected and also free of cellular debrises (vescicles or particles) whicha also may be present in cell culture. Only the soluble factors secreted by your original cultured cells are of interest in a culture supernatant. Centrifugation is therefore an essential part of culture supernatant preparation.
Thanks all. Would a centrifugal force of 10,000xg for 5mins at 4C be ok?
Probably yes, Generally yes, I would suggest you to comapre such centrifugal force with that necessary for spinning down cell lysate preparations (for mitochondria or endoplasmic reticulum, for instance: see in the literature): that way you may know what size of particles may remain in your supernatant and may possibly affect your results.
3500 rpm for 5 mins at 4 degree C is fine. It worked well for me (TNF ELISA). See this paper (Conditioned media=culture supernatant)
https://www.researchgate.net/publication/233710524_Smac_mimetic_enables_the_anticancer_action_of_BCG-stimulated_neutrophils_through_TNF-_but_not_through_TRAIL_and_FasL
Article Smac mimetic enables the anticancer action of BCG-stimulated...
Remeber that RPMs are only a trivial part of the story: depending on your centrifuge (radius and vials) a given G force is what you want to obtain for a given time.
Methods (see literature) call for given Gs for a given time to precipitate different size (molecular weight) particles. Follow centrifuge manuals and literature describing isolation of particles (organelles) you want to eliminate from your supernatant. This is the basic biochemical rule.
Hey Jhoti,
i did a lot of ELISAs and Multiplex Assays detecting different cytokines (of course TNFa too). I used serum-free medium or serum-reduced medium to cultivate my cells and for the collection of the supernatant. While collecting it i cooled the 1,5 ml tubes on ice, pipeeted the supernatant in and centrifuged them at 110000g 15 min and 4 degrees. Then i carefully transferred a bit more than half of the supernatant into new cooled-down tubes and put them in liquid nitrogen before store them at -80 prior to use. I was always able to detect TNFa in the supernatant.
when ur incubating the cells in ur medium ensure that minimum media required for sustenance is used....eg 2ml in 60mm plate......that will enrich the secretions...tnf alpha here....
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