I am currently pursuing a study regarding breast cancer cell lines. I'm looking for best/ simplest protocol for counting the cells.
Also, could you suggest machinery that would be necessary for said protocols? If you could, could you link me/attach a file as well?
Any advice would be much appreciated.
Hi Jason Tan Liwag
For seeding the cell for your experiment you can use trypan blue 0.4% solution do 10 fold dilution with trypan blue and count the viable cells using Hemocytometer
For cell viability experiment after experiment use MTT 5 mg/ mL solution from with add 10 micro liter for 100 microliter if you seed at 96 well plate and once the formazan crystal are formed use DMSO to dissolve it and read the color developed at 570 nm in a ELISA reader , and extrapolate the reading using the standard curve generated by seeding different cell density of your cells and incubated with MTT till the crystal development; from which you can have known OD for known cell density using this results you can easily get your cell viability/ count results post experiment
All the best
Trypan blue is the standard answer, but can be tricky if the cells are small, clumpy, low viability or faintly stained. If Trypan blue doesn't work reliably consider using 1:1 dilution with 3uM Propidium Iodide for 15mins @ RT, centrifugation and re-suspension, and a Flow Cytometric read with Ex ~488-535 nm and Em ~620 nm.
Best Trypan blue cell counter I've used is the Cedex HiRes, cheapest and easiest the Nexcellom. Easiest PI cell counter I've used is the NucleoCounter, which is safe and you'd have to work hard to muck up the counting.
Both Trypan and PI are toxic and known mutagens. If you want to preserve your cells' viability for use after counting there are some new dyes available for Life Tech, but I've not tried these yet.
hi
the easiest way is just using hemo-cytometer lams ... you only need to resuspend your cells in 1 ml of medium after centrifuging and put 10 uL of it onto hemocytometer lam, wait for 30 seconds then count cells under microscope ( you need to count all of cells on 16 medium squares) also it's better to count 2 or 3 other large squares, then multiply by 10*4 ... it's easy ... only takes 1 or 2 minutes.... it's easiest way but you know that there is better ones ... best regards
one more ... if you need to find dead cells you need trypan blue dye
I would agree with Martin Brown. Trypan Blue is a simple and cheap protocol only requiring a haemocytometer and a microscope. Automated cell counters are available but this obviously incurs a large additional cost.
I have recently added flow cytometry with Propidium Iodide staining to my cell counting protocol (also to allow discrimination of live vs. dead cells). As this protocol does not require you to count cells yourself a large element of human error is removed from the obtained values increasing reproducibility between samples.
That being said if all you want is a quick cell count before downstream experiments I would suggest that the trypan blue protocol would be the quickest and most cost effective for your application.
Best Regards,
Ruth
Most of these protocols are the same with insignificant differences. As for me, I dont stain the cells with trypan blue. I just believe if there are dead cells in the medium, the washing of the cells in the flask with PBS must have taken care of >90% of them and therefore, counting with staining with trypan blue might not be necessary. Maybe my method is not professional but it is time saving and also efficient. Cancer cell lines are easy to culture, I once worked with MCF-7. You dont need to stain while counting the cells. Just wash the cells 3 times in the flask, make sure you rinse the bottles dry of the PBS. The idea is that all the dead cells must be floating and if that is the case, they must have been removed by the PBS. So, when you count under the microscope on a Hemocytometer, you only spot out the viable cells. Then you can use your formula to calculate the number of cells and scale it down by diluting to soothe your seeding density.
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