I would set up the procedure of peritoneal washing to obtain rat resident macrophages (without previous recruitment and activation). We have had also difficulties in recovering adherent cells after 2h-adherence at 37°C. After detaching in PBS-5mM EDTA plus scraping, cell number and viability was very low (about 10% of the cells plated before adherence).
Can anyone kindly provide me with protocols and practical suggestions? Thanks in advance.
I used to cultured mouse peritoneal macrophages and also human monocyte-derived macrophages. They are really hard to detach, so I incubate them on ice for 30 min with PBS-EDTA. After the incubation I don't scrap, but I pipette vigorously so I can detach the cells with water flow with reduced harming. Also, how are you evaluating viability? If you are using a vital dye, macrophages are good to absorb them by phago/pinocytosis. I hope these suggestions help you a little bit.
Well there is low number of macroph., as resident, in peritoneal cavity so you'll get like 1ml of cells per rat.
As for detaching i suggest use Tripsin solution for cell cultures. Tripan blue dye is good for counting live cells after ;)
I hope i helped a litle!
Thank you all for your valuable suggestions.
I use Trypan Blue to check viability, and I have never used enzymes to detach.
Regarding the use of PBS-EDTA, my previous protocol suggested 30 min incubation at 37°C. Do you feel that ice-cold incubation is better to detach?
Thanks a lot again
Since macrophages are good in uptaking particles, some cells will probably absorb trypan blue even if they are not dead and permeable. So, its better to be really quick when using trypan blue with macrophages. Also, you can check viability using MTT assay or flow cytometry with propidium iodide. Coupling both PBS/EDTA and low temperature will certainly enhance the detachment, avoiding the use of enzymes (they are expensive and can reduce viability too). However, you need to help the detachment with vigorously pipetting. I agree with others, trypsin could be used and cells will detach easily.
I understand: in this case, my cells could be "false" dead! I can check viability by MTT assay, but the problem is that I need to count cells after recovery, to evaluate the yield from each rat for consistency and standardization purposes. Perhaps, I could use a nucleus-staining dye...
I do agree with Vitor De Rezende..For detaching macrophages (mouse peritoneal macrophages) I used to use ice-chilled PBS for 15-20 minutes on ice and then pipetting...with scraping there is a chance to get lesser number of viable cells..
Thanks! I followed a similar indication (ice-cold PBS-EDTA 2mM for 30 min) in a new experiment and I recovered about 30% of live cells.
For cell counting you can use older method and stain them with Turck solution (easily made) and it stains macrophages nice :)
You wont get false dead cells because the method is based on dye uptake by cells...so only live cells can uptake it :)
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