I am trying to carry out trypan blue assay. However, when I did the experiment, I was so confused about what is dead cell.
My protocol : I cultured MDA MB 231 cell lines in plastic dishes. After treating cells with trypsin, I diluted them with media, then mix cell mixture with trypan.
1. When I observed haemocytometer, I saw a lot of blue fragments ( its size is 1/3 1/4 of normal cell) and blue dots. So I don't know should I ignore them or I have to count them? Do I need to care about cell-sized objects.
2. Because there are too many cells, I diluted cells with trypan with ratio : 1: 3. Would it be ok?
3. I thought if the cell concentration is too low then counting would not be accurate. Do we have any minimum concentration of cell?
4. Because I used trypsin to detach cells, I centrifuge them and removed trypsin and media. However, the amount of cell is low ( I use 6 wells-plate) so suction remove a lot of pellet? What I should do to tackle this problem?
Please help me. Thank you so much.
There are few points I want to make clear, such as-
- The dilution should be 1:4.
- The cells with blue cytosol are dead cells, because viable cells do not allow trypan blue to enter inside the cell cytosol.
- One has to count both blue cells and clear cytosol cells separately to calculate % viable cells.
- Follow link below for more precise protocol and video.
Thank you!
http://www.abcam.com/protocols/counting-cells-using-a-haemocytometer
1. Trypsinize cells (1ml/well , keep an eye on them so you don't cause cell death (can use 0.05% if 0.25% is too harsh). Add media with 10%FBS to inactivate trypsin. Spin 1500 RPM x 5min. If suction is too vigorous, try pouring off media. Resuspend in media.
2. Add 50ul cell suspension + 50ul 0.4% trypan blue, mix and add 10ul to hemacytometer (if using Bright line or Hausser type glass hemacytometer with glass coverslip). Count # cells (blue and clear) in all 16 wells in each quadrant and take the mean. Multiple by 10,000 x 2 (for dilution)=total # cells/ml. Viability = Total #cells minus #dead cells (blue) divided by the Total. You should have 90-95% viable cells.
3. If cells are too numerous to count, dilute them first (1:10 or 1:20, etc) and then repeat the 50ul + 50ul mix. Just remember to multiple your total by the dilution factor (10 or 20)
Good-luck
http://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/trypan-blue-exclusion.html
Thank you for your answers but I still have some confusion.
1. Actually, I don't care about cell concentration. I just care about cell viability. I am wondering if my sample has too low concentration of cells, I would not reflect the exact viability. Is there a minimum concentration.
2. Should I ignore blue dots which are negligibly small than usual cells?
3. I observed cells which have blue cytosol and clear nucleus and cells which have clear cytosol and blue nucleus. How can I rate these cells?
Thank you once more time.
I have never seen what you describe. Can you try a fresh bottle of trypan blue? Is it homemade? Maybe it is contaminated?
Cells cultured in a 6MW plate should produce plenty of cells even if 50% confluent.
Try with fresh reagent.
Live cells look birefringent on a lighjt blue background when you mouve the micro
Take a drop of your reagent without a cellsuspension under your microscope and you will know if the dark small dots are coming from it or not! If yes, you can warm up your reagent or filter it through a 0,2mM sterile filter to be able to use it again. If not, perhaps you are disturbing your cells by trypsinisation or centrifugation ( 200g is enough to pellet the cells)
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