Hello, I am learning how to isolate thioglcollate primed peritoneal macrophages from mice but my yield has been lower than expected. I expect a total yield of 1x10^7 but I have had inconsistent and sometimes very low yield (ex. 3.2x10^6 cells in a recent periwash).
My first problem has been trying to aspirate the media from the peritoneum. I have improved on that method but I still unexpectedly had the yield above. Is it possible that the cells failed to infiltrate the peritoneal cavity?
I have outlined my steps below. Would readers familiar with this method be able to review my steps and suggest some sources of improvement?
1. Inject mouse interperitoneally with 1 mL 4% thioglycollate
2. Wait 4 days for cell infiltration
3. 4th day Inject 7mL media (RPMI+2%FBS)into peritoneum with a 27g needle.
Massage cavity gently.
4. Carefully use a 25g syringe to extract the fluid from the cavity with a yield of ~5ml
5. Dispense cells into a tube placed in ice
6. Spin cells for 10 min at 300xg, discard supernatant and resuspend cells in 1-5mL media(RPMI/2%FBS/Pen/Strep/L-Glutamine)
7. Cell count: Need 2x10^6 cells/well in a non-tissue culture treated 6 well dish
8. Incubate in 37*C,5% CO2 overnight
9. Next day-remove media from cells and gently wash each well with 1mL room temperature sterile PBS
10. Add 1mL ice cold 2mM EDTA in PBS to well
11. Sit plate on ice for ~30 minutes, then gently tap the plate on a hard surface
12. Draw 1mL EDTA up and down to gently dislodge cells and collect.
13. Spin cells for 10min at 300xg, discard supernatant and resuspend cells in 1mL media
14. Count cells-add to untreated coverslips in 24 well plate. Let cells adhere to coverslips for ~20mins at RT
I am a beginner to cell work and specifically with macrophages so any insight would help!
Thank you!
Dear Janna Bashar
I briefly wanted to reach out concerning your question.
Did you consider aging your thioglycolate preparation (for a month or so) as per the publication below.
Article Glycation products in aged thioglycollate medium enhance the...
Moreover, you can consider polyacrylamide beads (biogel) elicited macrophages, which should have higher yields.
Please check the attached protocol.
http://www.bowdish.ca/lab/wp-content/uploads/2014/03/Harvesting-BG-pMøs.pdf
The peritoneal macrophage populations will differ slightly functionally, which may impact your downstream experimental setup.
For a side by side comparison check out:
Article A Real Time Chemotaxis Assay Unveils Unique Migratory Profil...
I hope that these considerations help.
All the best & good luck with your experiments,
Michael
Few things to consider....
1. Thioglycolate that has been aged at least for a month will give a better result. The older, the better.
2. During recovery process, inject 10mL of ice-cold PBS into the peritoneal cavity using a 10mL syringe with 25gauge needle and you would get about 8 ml back. You may use PBS containing EDTA 2 mM to avoid cell clumping which in other way increase the yield.
3. Importantly, after seeding the cells to the 6 well plate, try to change the media after 2h of seeding. Cells usually get stuck by then and any non-adherent/diseased/dead cells will be washed off.
4. Looks like ur interested in finally getting the cells on to cover slips. In that case, better you initially seed the cells in wells with cover slips in. This will avoid using EDTA for dislodging the cells, and the time to re-seed them on cover slips, which altogether may affect the viability/integrity of the cells.
Otherwise, the protocol which you are using is absolutely fine.
BW
Hello, thank you for your responses.
My thioglycolate has been aged after purchase. But I will revisit if I have missed something related to it's condition.
Michael Förster Thank you for the article suggestions.
Vinod Nadella I hadn't considered using EDTA initially to increase yield-thank you.
I do use a 25g to aspirate but it sounds like I could increase my yield by increasing the volume.
For your #3 suggestion. So you are recommending doing a 2 hour incubation instead of a overnight? Or just include a 2hr step to freshen up the media?
Your #4 suggestion is something I will consider, thank you.
I mean, just change the media to remove any unadhered cells and then continue to incubate ...
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