I have co-cultured dendritic cells with T. cruzi and I need to evaluate the cytokines realized from this interaction. How can I harvest and storage the supernatant after the infection to perform ELISA assay and how is the recommended method to this assay?
Hi Amanda,
Areeful is right, but I guess that depending on how much supernatant you have you may not need to spin it down for that long. Having said this, if your cells after incubation have been transferred to eppendorfs, I guess you may centrifuge these at 5000 rpm for 5 min in a microcentrifuge. Then, you just need to remove the supernatant to a fresh eppendorf and store these in a -80C freezer. You can still keep the cells for further analysis if you wish.
I hope this helps! Boa sorte!
Hi Amanda,
Areeful is right, but I guess that depending on how much supernatant you have you may not need to spin it down for that long. Having said this, if your cells after incubation have been transferred to eppendorfs, I guess you may centrifuge these at 5000 rpm for 5 min in a microcentrifuge. Then, you just need to remove the supernatant to a fresh eppendorf and store these in a -80C freezer. You can still keep the cells for further analysis if you wish.
I hope this helps! Boa sorte!
Your dendritic cells are, probably, adherent. So, it's easy to harvest supernatant from the wells (even without spinning), and then centrifuge it in the eppendorfs, as Diogo said. Most of your cells will be still on the plate.
To save handling and freezer space, we often freeze directly into 96 well plates (chose low absorption ones where possible). That way you can transfer samples easily into the ELISA plates once defrosted and also quickly and easily label (or same a separate lid key). However, you can't store samples like this forever (a few weeks is fine), so it wouldn't be ideal for long term storage. It depends a bit on how long you need to store them for and how many samples you have.
Amanda, is there any difference between flat or round-bottom plates for storage of supernatants?
Carolina - I generally use round bottom plates for storage as you get a greater recovery of your supernatants, as the liquid pools nicely at the bottom of a round well rather than being lost to the sides of the flat bottoms where it is harder to pipette. Other than that there is no major difference (just check you are using low adsorption plates and not tissue culture ones!)
Hi
I don't have any microcentrifuge with low temperature, can I centrifuge my sample in room temperature?
my medium include 20% FBS.can it affect my result?
Hi Mina,
It depends on what you are looking for?
If you prepare a cell lyset from your sample, and you already used the lysing bufer (eg. Ripa) if you have enough amount of protease and phophotase inhibitor in it, it would be fine to centrifuge your cold sample for like 10 min at room temperature.
another thing is you can put the centrifuge in the cold room overnight and then its gonna be more safe n relax to centrifuge your sample.
and if you wanna centrifuge living cells to get the cell pellet, it is still fine to do it up to 5-7 min without cold temperature. however keep in mind it is important to keep all condition the same to repeat the experiment and reproduce the result again.
Can I store the supernatant in -20 degree freeze or it needs to be stored at -80 degree?
Hien Nguyen It is recommended to store the supernatant at -80 degree in aliquots and avoid "freeze-thaw cycle" for longterm storage (up to 6 months or more). I used to freeze my samples at -20 degree for 1 month and TNF-alpha, IL-6 ELISA assay with those samples still worked well in my experiments.
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