Preparing Conditioned Media Samples for ELISA Kits


RayBiotech manufactures over 1,000 high quality, rigorously tested ELISA kits. In this blog post we explain how to prepare conditioned media samples for your ELISA Kit.

How to Prepare Conditioned Media Samples

We recommend preparing serum-free or low-serum medium samples, as serum tends to contain cytokines which may produce significant background signals. If it is necessary to test serum containing medium, we recommend also running an uncultured media blank to assess baseline signals. This baseline can then be subtracted from the cultured media sample data.

  • On day 0, seed ~1 million cells in 100 mm tissue culture plate with complete medium.*
  • On day 3, remove medium and replace medium with 6-8 ml of serum-free or low serum containing medium (e.g. medium containing 0.2% calf serum).
  • On day 5, collect medium into 15 ml tube. Centrifuge at 2,000 rpm in centrifuge at 4ºC for 10 minutes. Save the supernatant. Transfer the supernatant into 1.5 ml Eppendorf tubes. Store supernatant at -80ºC until experiment. Most samples can be stored this way for at least a year.

*The optimal number of seeded cells varies from one cell type to another and may need to be empirically determined.

Note: In case follow-up experiments are needed, it is strongly recommended to sub-aliquot all samples after preparation to minimize cytokine degradation from multiple freeze-thaw cycles.

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