It seems that the method of cell counting with a TC20 cell counter by Bio-Rad is associated with with high variations. Therefore, I am searching for a better method to improve the accuracy. Can anybody suggest a precise method for cell counting or recommend a paper where the different methods were compared?
I would like to count neuron-like cells in a collagen-coated 6-well plate.
Thank you for your help, Alisa
An easy and precise method to count cells in cell culture plate is taking pictures with an Inverted microscope (like Zeiss' Axio Observer A1 ) and analysing the image with an image analyser software (Zeiss' Axiovision, ImageJ, ImagePro or others). All of them have plugins and procedures for cell counting.
I use a similar instrument from Life called Countess. dont know how they compare but there are other methods that are independent of trypan blue and use DAPI for example you might want to look into those.
we usually employ different methods for cell counting. I assume you are looking for proliferation. If its for proliferation then we do it in 3 different ways
1) By trypan blue staining 2) WST 1 assay 3) mts assay.
Dear Jair,
Counting cells that are highly irregular (e.g. neurons) is difficult either manually or by an automated method. In my opinion the best would be to stain nuclei with DAPI and count either manually with a fluorescent microscope or , after taking a frame, by any reasonable software (e.g. Metamorph) after thresholding and converting to binary image. If your support is blocking UV, there are 96 clusters designed for UV. If you do not have access to fluorescent microscope, you can count nuclei on a regular H&E staining, but only manually since no software is good enough for that. If you do not want to kill the cells, you can use Hoecht's stain, which is supravital. You'll stil need fluorescent scope for that.
Hope that helps
The question nis really not clear to me. Do you need to count live cells and then culture them afterwards? Or, you only want to figure out how many cells on the plates with limited surface area. The method will be very different according to your purpose. Using TC20 counter you have to make sure that the cells are really separated from each other otherwise multiple cell may mistakennly count as one. If only limited number of cells on a small surface area then DAPI is not a bad method but you can not use the cells afterwards.
Dear all
Thank you very much for your answers. I am using differentiated SH-SY5Y cells in an functional turnover assay and have been counting them afterwards (TC20 after trypsinization) in order to obtain comparable results. However, the TC20 doesn't seem to be very precise and therefore I am looking for an alternative method.
Your suggestions are great, unfortunately my cells are not evenly distributed throughout the plate, therefore I have been using a method where I trypsinized them beforehand.
To specify my question further: is a Coulter counter probably more precise? Are there any other automated cell counters? Or is the the manual hemocytometer still the preferable method?
There are other cell counters out there. Nexcelom makes a variety of imaging cytometers that will meet your needs. We have sales reps in Europe who can provide an in - lab demo for you.
Our website: Nexcelom.com
bill
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