Currently I am plating human monocytes at a concentration of 1million/mL in a 48 well-plate where I add 400,000 per well. I differentiate them by adding MCSF (100ng/mL) for 6 days (change media on day 3) and on day 6 I use specific conditions (ex: LPS and IFNG). My differentiation has worked based on qPCR results (where I lyse the cells directly on the plate and isolate RNA from that). However, for phenotyping at the protein level I need to do flow cytometry and my recovery has not improved over the last months.
Currently what I do is:
1) Remove media and add cold PBS+5mM EDTA and incubate 5 minutes in ice. Then I add Accutase for 15 minutes at 37 degrees, then neutralize with same amount of warm R10 and wash with cold PBS +5mM EDTA. Then I add again Accutase but leave at room temperature for 15 minutes. Then wash again with cold PBS +5mM EDTA.
2) Add 200uL of cold PBS +5mM EDTA and scrape the cells from the plate and spin at 900g for 5 minutes at 4 degrees. (I use this speed as at lower speeds the recovery was barely 20%, as soon as I increase it the recovery increased drastically).
So far I have found suggestions about leaving on cold PBS or in the fridge but it has never worked and cells barely detattach. I also tried Trypsin but did not work and I found many recommendations on not using it as it affects the expression of surface markers. This approach has gave me the highest recovery (up to 50% back).
My questions:
1) Should I use Accutase differently?
2) Should I increase the concentration of EDTA ?
3) Should I increase the time I leave the cells in the fridge on PBS + 5mM EDTA?
4) Should I change the conditions for the final spin?
Dear Juan Diego Sanchez
I have also worked on MDMs and for RNA and Protein i used cell scrapper as morphology does not impact for those two things.
But for characterization, one needs the cells to be intact. I earlier tried with accutase (as trypsin was causing surface protein deterioration) for around 30 mins at 37 degree with intermittent palm tapping after washing with cold PBS. It worked for me.
You must try with different time points and increase agitation. Do check cells under microscope before placing these in tube.
Another thing you may do; grow MDMs on coverslip and perform microscopy for surface markers.
Wish you all the best.
Hi Juan,
I have this issue now for months working with primary Monocyte derived macrophages and here is my advice for flow cytometry analysis:
Accutase detachment takes much longer than 15 min if you use regular cell culture plates. For me cells start detaching after 30min Accutase at 37°C up to 1h depending on the polarization used. Vigorously clap against the plate and check every 10 min for the detaching - be patient. The polarization markers I was looking at (CD206, CD209, CD40, CD38, CD163) were still delectable in flow after prolonged Accutase treatment- so give it a shot for your cells. Viability was fine after prolonged Accutase treatment.
Alternatively if you absolutely do not want to use enzymes, I can recommend using the Thermo NUNC UpCell plates in combination with macrophage detachment solution from Promocell. You wash your cells with PBS and leave them with this solution on ice for 1h. They start to come of then with good viability.
PBS+EDTA never worked for me either. I don´t scrape primary monocytes since the viability is reduced to only 30% viable cells in flow analysis.
Another comment: You seed a lot of monocytes into the 48w formate. For qPCR this makes sense since you want to get as much RNA as possible. However, I found that seeding them too densely makes it difficult to detach cells. I use 1 Mio cells per well of a 6w plate for example :)
Hopefully these ideas help your research.
Best,
Bianca
We always use scraping to harvest cells for flow cytometry as use of trypsin EDTA may have affect the expression of cell surface molecules. Macrophages adhere to cell culture plate firmly so you should try 0.5% Trypsin EDTA at 37 C to harvest them. It always works in our hand.
Hi Juan,
Why are you using cold PBS here? To avoid loss of surface markers?
I don't think cells are so sensitive to surface marker loss. Have you tried using TryPLE which is milder than Trypsin? I am not familiar with macrophages but stem cells don't ever lose surface markers by TryPLE treatment.
I see you are making a mistake at your initial step. You are incubating cells with cold PBS+EDTA. How about washing cells with PBS before that? Ideally I would wash cells once with RT maintained PBS and treat them with accutase at 37 degree for whatever duration is ideal for your cells (30 min in this case as suggested above). Tapping the plate is a good idea at the end but once you see cells getting rounded. Also, once cells become rounded, manual pipetting should scrape cells off from surface without killing them. I won't suggest scrapping the cells.
Hope it helps!
Hi Juan
I have been using cold PBS with protease inhibitor (1 tablet) in 10ml PBS but previously in my lab people have also used Accutase enzyme and I think it will be easy to use it in the plate as compared to scrapers.
Good luck
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