Troubleshooting: The Cell Count is Incorrect

Modified on Mon, 23 Oct 2023 at 04:18 AM


My cells are not detected successfully, leading to an incorrect cell count. 


Improper cell detection can be caused by an incorrect focusing technique. The artificial intelligence algorithm is trained to recognize a lot of different cell types and can even detect individual cells within cell clumps. Nevertheless, when the focus is not correctly set, the detection can fail. Ensure that the cells have a white center and distinctive black edge. Read more about cell focusing here: Helpdesk : Axion BioSystems, Inc. (freshdesk.com)

There are a lot of bubbles in my sample.


Bubbles can be recognized as big black circles. Bubbles can lead to false results, as they influence the distribution of cells and the volume inside the counting chamber. The cause for bubbles is often due to incorrect pipetting. Therefore, try to prevent having air bubbles in your sample and prevent air uptake during the loading of the sample into the chamber.


There is a lot of debris in my sample.


Debris may be caused by improper cleaning of the hemocytometer. To resolve this, properly clean the hemocytometer with wet lint-free wipes and load the sample again. Please note that cleaning with water is better than with ethanol: ethanol is a fixative and can fixate the previous sample to your reusable counting slide, increasing the cell count.


I used the cell counter to determine my cell concentration and compared it to a manual count, but the cell counter gives a much higher cell count. 


There are a few things to check when performing a cell counting experiment, and comparing it to a manual count:

1. Download the latest version of the app from download.axionbio.com. Any combinations of sample, cell detection and calculation factors that we noticed didn't work optimally before are solved in the newest version.
2. Make sure you are using a counting chamber of 0.1 mm high. There are also 0.2 mm high counting chambers - mostly used for organoids - but these give results that are a factor 2 off.
3. Count exactly the same regions in the counting chamber. For the fairest comparison, use a hemocytometer with grid, and count the same squares in the hemocytometer grid manually and with the cell counter.
4. Confirm that the objects detected by the cell counter are indeed cells: check the images with the overlay of detected cells. If debris is detected, change the size gating sliders. Ensure correct focusing.
5. Make sure the dilution factor is set correctly. The cell counting results already consider the dilution factor to calculate the concentration in the original sample, so it's not necessary to multiply results by the dilution factor again. Trypan blue counting assumes a 1:1 dilution, but requires no extra dilution factor to be included. Read more about the cell dilution calculation here: Helpdesk : Axion BioSystems, Inc. (freshdesk.com).   


 


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