Improving Cell Counting Accuracy

Suspend to single cells:
- Gently pipette the sample to suspend the cells evenly before counting to avoid clumping.
Wash sample:
- Wash the cells to remove any debris.
Optimize concentration:
- Obtain the proper range of cell concentration based on the counting method:
  - Typically, 10⁴ - 10⁶ cells/mL for hemocytometers.
  - Follow the recommended concentration range for the counter you are using.

Calibrate tools regularly:
- Automated cell counters and pipettes should be regularly calibrated for accuracy.
Clean optics:
- Clean the objective lens of the microscope or make sure no sample is spilled on the lens of the counter. This helps provide a clear view of the cells and prevents miscounting.

Choose the right staining dyes:
- Use Trypan Blue for manual counting or brightfield-based counters to distinguish live and dead cells.
- Use fluorescent dyes (e.g., Propidium Iodide, Acridine Orange, or DAPI) to stain the nuclei, which allows for more accurate distinction.
Follow the recommended staining time:
- Overstaining may reduce cell viability due to dye toxicity.

For Automated Counters:
- Adjust parameters such as cell size range (size gating), circularity (cell roundness), brightness, and focus if applicable.
  - Size gating and circularity help exclude unwanted substances like debris.
  - Proper focus is critical for accurate counting.
For Manual Counting:
- Avoid double-counting cells on gridlines by following standard counting protocols. Click HERE to see detailed protocol.

Take multiple aliquots of the sample:
- Take multiple aliquots to prevent uneven distribution. Thoroughly suspend the sample before obtaining aliquots.

Consistent cell counting:
- Establish clear criteria to differentiate live and dead cells to reduce user variability.
- Users should be trained to distinguish cells from debris.

Cell focusing:
- For counters requiring manual focus, follow the manufacturer's guidelines for optimal results.
Optimize Imaging Settings:
- Adjust parameter settings such as brightness and calibrate regularly for accurate detection.
Use compatible fluorescence dyes:
- Each fluorescent cell counter uses specific optical filters with different optimal wavelengths. Use dyes provided or recommended by the manufacturer for the best results and image clarity.