I am performing an experiment analyzing the cytokine production of macrophages in response to viral infection.
I recently harvested and cultured peritoneal macrophages from mice. I plated these cells on 12-well plates and have been changing the media enriched with 1:1000 mCSF every two days. Once the cells reached 80-90% confluent I washed the cells two times with PBS and infected these cells with an inoculum of virus at an MOI of 3. After 1hr incubation, I washed the cells again two times with PBS and put 750uL of media back on cells. After 24hrs, I collected this 750uL of media to be used for 36-spot Luminex.
I'm concerned that I placed too much media on the cells after infection - do you think I have diluted the supernatant too much and my cytokines will all be below the limit of detection when I run a Luminex assay?
Thank you for your help.
Olivia
You have not told us the problem you encountered, only your fear. I do not know what the problem is and so cannot tell where it lies.
Hi Maurice Ekpenyong , I haven't encountered a problem yet with this experiment, but I was wondering whether others had done a similar type of experiment and knew what the optimal volume was for collecting cytokines in the supernatant of a 12-well plate for a Luminex assay.
hi Olivia B. Parks,
I am not familiar with these kinds of macrophage stimulation experiments, but I have a background with other non-immune cells. If I were you I would consider some points in minds to guarantee satisfactory results:
1- Check the datasheet of your Kit, usually important information is there. The manufactures do their best to keep everything clear about their products, If any queries you might find contact data for technical support. Give them a call or send an email.
2- Scan literature used the same Kit. Read their material and method section very carefully and try to expect if you are moving on the right pathway or not.
3- A senior colleague who has used the same kit with the same type of cell is another good reference,
Best of luck
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