I differentiated monocyte- derive macrophage on coverslip in 24 well plate with 1x10^6 cell/ml in total volume 1 ml per well but some cells died after seeding.
I have washed non-adherent cells at 1 day after seeding. Then I've been culturing macrophage until day 7 and the confluent cells is about 70-80%.
I've tried to collect macrophage by scraping and using EDTA and Trypsin but it make macrophage died and some cell were getting defragment. Cell number lost , so I'm trying to culture them on coverslip
My point is that I need to know the number of cell. How I estimate them? before I infect them with virus
Can I count cells under microscope by taking photo and calculate the number from magnification or calculate by using a size of macrophage divided by area of well (24 well is 1.9 cm2)
Does anyone know the size of macrophage when it adhere on plate?
Thank you very much
You can get certain valuable informations by following the links given below:-
1. http://www.sciencedirect.com/science/article/pii/S0304401713004779
2. http://jgv.sgmjournals.org/content/journal/jgv/10.1099/0022-1317-83-4-747?crawler=true&mimetype=application/pdf
3. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071098
I have a suggestion. What if you proceed with viral infection, then do immunofluorescence staining using anti-virus, and anti-macrophages with/without anti-monocytes. I attach here a previous discussion on which markers best to differentiate between human monocytes and macrophages. All the best!
https://www.researchgate.net/post/Which_markers_are_best_to_differentiate_between_human_monocytes_and_macrophages
I used a microscope micrometer ruler by taking a photo and compare area in picture between the ruler and culture-plate. It's fine. I finally estimated and calculated a number of cells. I'm not sure my method is appropriate or not but that's the only way I could fix my problem. Thank you for the comments
It looking well if you want to estimate density of cells. It may be sufficient for your purposes (some fluctuation of density could account to over and underestimation).
Optionally it possible to estimate metabolic activity of cells and calculate "normolized" quantity by calibration curve with known number of your cells (be sure what stimulation and ROS production don't affect your assay).
You can also try using non-tissue culture treated (but sterile) plates for your culture. It's very hard to detach macrophages from TC treated plates. Also, to help them detach put them on ice for about 30mins with EDTA ... it might help in detaching the macrophages.
It is very hard to detach macrophage if you seed bone marrow cells on 24 well plates during differentiation. I usually differentiate them in 150 x 15 mm sterile petri dishes (Falcon) for 6 days. Basically, for each mouse femur/tibia, I put 20ml complete media and 1/5 fresh media is topped up each of 2 days. At day 6th, I remove media and wash once with warm sterile PBS to remove non-adherent and dead cells, then put 10mL of 1mM EDTA/PBS and incubate cells in 10min at 4oC. When collecting cells, I put additional 10 mL cold PBS to pipette cells out of the plate surface, then another 10mL PBS to rinse the remaining cells. After that I spin cells down, resuspending them with fresh media, counting the total number of cells and plating them on 24 well plate/coverslips for experiments on day 7th. The number of cells would not change much after differentiation. Choosing dish to differentiate macrophage is important, as different dish has different adherence properties that could not detach cells easily with Tripsin or EDTA. The 150x15 mm of Falcon company worked for me.
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