I want to expose my lung epithelial cells to different treatments in vitro and then detect pro- and anti-inflammatory cytokines/chemokines in the conditioned media - can I then do PCRs on the conditioned media? Any guidance would be very much appreciated!
PCR is used to detect nucleic acids, not proteins. Generally, you would use either a Western blot or an ELISA to measure the amount of a secreted protein in cell culture media.
Hello Atefeh Mohammadi
Cytokines/chemokines are small, secreted proteins released by cells that have a specific effect on the interactions and communications between cells. These proteins can be best detected using ELISA. ELISA is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. In brief, in ELISA, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then complexed with an antibody that is linked to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. In your study, ELISA will be the most appropriate technique to detect cytokines/chemokines in the conditioned media.
On the other hand, PCR is a technique used to make numerous copies of a specific segment of DNA quickly and accurately. In your study, you cannot perform PCR because you are interested in proteins in the conditioned media and not DNA.
Best.
hi dear , besides the RT PCR of target gene expression , you can consider Immunohistochemistry or Western blot to evaluate the cytokines/chemokines. Sometimes it is good to combine at least some of those methods to emphasize the consistent results. hopefully, this sent protocol can meet your question comprehensively.
Hey Atefeh Mohammadi ,
to detect cytokines and chemokines I would recommend a multiplex ELISA platform like LUNARIS where you can detect multiple proteins at the same time.
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