Cell Counting Support—Troubleshooting

Exposure to light may degrade the dye and these contaminants may promote precipitation. Trypan blue can also form orange/red fibrous aggregates if exposed to refrigeration or freezing temperatures. 

Unfortunately, there is no definitive answer to this question. The rate at which the cell-impermeant dye is absorbed depends on the cell type, their state of health, nourishment, engulfment activity, etc.  

The most recent version of the Countess™ II or Countess™ II FL software is available here. Always check the website to see if a new software version is available if you are having any issues since the software is updated regularly. You can also register your Countess™ II or Countess™ II FL instrument here to be informed of any future software updates. 

The brightness and size sliders establish the thresholds for the imaging algorithm, allowing users to set both the brightness and size ranges for the sample. If the parameters are at their maximum settings, all brightness levels and sizes are counted, so they cannot be further adjusted. To narrow the brightness or size range for the count, you can move the ends of the slider bar or you can hit the icons at the end of the slider bar to adjust them. To demonstrate this while on the ‘Adjust’ screen, repeatedly tap the ‘dim’ icon on the brightness slider or manually move the bottom end of the slider and watch the green brightness bar decrease in height. The resulting narrowed brightness range can be slid across the brightness scale to include only dim cells, only bright cells, or something in between depending on what is to be included in the count. The same adjustments can be performed for cell size.

Select the “Default” profile to restore the size, brightness, and circularity gates to their maximum.

Uneven illumination on the screen may be fixed by resetting the light cube tray. First, make sure you have the most recent version of the Countess™ II or Countess™ II FL software installed on the instrument (it is available here). If the software is updated, the light cube tray should readjust each time the instrument is power cycled. You can also select the “change light cubes” option under the settings menu, allow the light cubes to reset, and then turn the instrument off and back on. This process may help with uneven screen illumination even if you do not have light cubes present or if you have the Countess™ II instrument (without the fluorescence option). 

First, make sure you have the most recent version of the Countess™ II or Countess™ II FL software installed on the instrument (it is available here). You may need to manually adjust the focus and set the nominal focus so that the autofocus will have a set point to focus on the cells. Once cells are stained 1:1 in 0.4% trypan blue and placed in the slide, you may want to let the cells settle for around 30 seconds. Then place the slide in the instrument and after you let the machine autofocus, you may need to refine the brightfield focus with the manual focus. Ideally the live cells should be slightly under focused, where the centers are bright compared to the dead cells that are dark throughout. You may want to zoom in so that you can better see the cells, then manually adjust the focus using the focus slider bar before you hit capture. Once the cells are focused, you can tap the blue “set” box to set the nominal focus; setting the nominal focus will improve autofocus consistency with future slides. 

Make sure there are no bubbles or debris visible on the screen that could interfere with the autofocus and make it more difficult to get the sample in the correct focal plane.

You can find some more guidelines for focusing starting on page 20 of the manual.

Decrease the brightfield and fluorescence light intensity before counting the cells. 

Once you press “Capture” to count the cells, hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. It’s best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness. 

You can also select the “Default” user profile to restore the size, brightness, and circularity gates to their maximum.

If the gates are fully maximized, the CSV should indicate 0–60 for cell size min and max and 0–255 for brightness min and max for both live and dead cells. 

The screen has a thin plastic film screen protector that should be removed before use.

The light source adjustment controls the LED intensity, camera gain, and exposure time, and it is used for adjusting the image brightness and contrast before you capture the image. Once the cells are inserted, there will be a bar on the right hand side to adjust the light source intensity. Often it helps to turn it down so that the background is slightly darker, which allows better contrast between live and dead cells. Optimal light intensity in brightfield is usually around 10–20 if there are no light cubes in the instrument, and slightly higher around 20–30 if there are light cubes present, but this could vary depending on the sample.

Once the image is captured, you can also adjust the range of cell size and cell brightness that the counting algorithm will count. This doesn’t change the actual brightness of the image or how it looks, but determines which cells will be counted. Once you press “Capture” to count the cells, you can hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized by maximizing the slider bars to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. This is mainly an issue if there are cells that appear in the image, but aren’t being counted. It’s best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness.

The brightfield cell concentration assumes that you have diluted the cells 1:1 in trypan blue, so it already takes this dilution into account and multiples the values by 2 so that the cell concentration displayed is the original cell concentration before dilution in trypan blue. The total cell concentration displayed after a fluorescent count does not take any dilution into account, so it is the actual cell concentration in the slide.

Ensure that a FAT-formatted USB drive is inserted in the instrument.

If you are pipetting different samples from the same cell sample, the variability could be due to pipetting or mixing. Use recently calibrated pipettors and make sure that the cell suspension is well mixed by pipetting up and down several times before you add 10 µL to trypan blue. Once the cell sample is mixed with trypan blue, it should also be well mixed by pipetting up and down and loaded into the Countess™ slide right away.

If you are counting replicates of the exact same slide, visually inspect that all cells are counted correctly in the image. Ensure that you do not shake or agitate the slide between counts. There may be a slightly different field of view each time a slide is inserted, so depending on the concentration and uniformity of the cells, there will be some variability when performing replicate counts of the same slide, although it should be less than 10%. When counting fewer cells, a small field of view change for only a small number of cells can have a larger affect, so variability can be reduced by counting cells at a higher concentration.

There are two predominant reasons for this: either the cells in question are truly dead or the focus should be adjusted. In some cases, heavily pigmented cells will also look dead in brightfield; such cells may require fluorescent viability stains.

Ensure that the cells are focused correctly so that live cells have bright centers and dead cells are dark throughout. If the cells are not well focused and look dark on the screen, the Countess™ II or Countess™ II FL instrument will count them as dead cells. If cells are well focused, have bright centers, and are being counted as dead, confirm that they are within the appropriate cell size range and try adjusting the settings. If cells are exposed to trypan blue for a long period of time, viability could be affected so the slide should be prepared and counted fresh each time for best results.

In some cases, heavily pigmented cells will also look dead in brightfield; such cells may require fluorescent viability stains.

• The drive must be FAT32 formatted; verify this before proceeding. At present, you should assume that all other formats will not work. 
• The update file must sit on the top level of the USB drive, not within a folder or subfolder.
• File cannot be renamed in any way.
• File cannot be zipped or compressed during distribution. It must be uncorrupted during transfer. 
• The device takes a few seconds to recognize the USB drive.
• If needed, check that the USB port is functional by testing a USB mouse.

To format a USB to FAT32:

1. Insert drive requiring reformatting into a PC, find dive in “My computer”
2. Right click on USB drive, select Format.
3. Select file system, FAT32.
4. Click Start

Reformatting will result in the loss of all files from the USB drive, so ensure that you have copies of any important files on another drive prior to reformatting. After reformatting, the software should be retransferred onto the USB drive prior to updating the Countess™ II or Countess™ II FL software.

The Countess™ II or Countess™ II FL instrument can also be controlled by a USB mouse, so you can try reinstalling the software by plugging a USB mouse into either USB port on the instrument and use the mouse to perform the software update.

The original Countess™ Automated Cell Counterhas been discontinued. However, we still offer consumables for the instrument. The most current software and firmware for the original Countess™ Automated Cell Counterare still available here at the bottom of the page under “Original Countess Software Download”. The software is also provided on the USB drive that comes free with the instrument. This software allows you to manipulate your cell-counting data and to save your data in a pdf report format for printing or archiving.

Note: As an alternative to the original Countess™ Automated Cell Counter, we offer the Countess™ II and Countess™ II FL Automated Cell Counters.

When the original Countess™ instrument freezes after a count, this indicates that the .csv file is full and should be erased. The .csv file can only hold 500 lines of data, so depending on how much you use the instrument, you will need to periodically clear the .csv file so that it does not freeze after a count. Here is the protocol for clearing the .csv file. Remember to save any data from the CSV that you want to archive to a USB drive before clearing the .csv. 

1. Perform a Count without the slide
2. Press Save button
3. Press View .CSV File button
4. Insert USB drive and press Save to USB
5. Press Close button
6. Press Start .CSV File button and the following prompt will occur: “This action will erase the previous .CSV file and start a new one. All data files saved will now be added to the new .CSV file.” 
7. Press Continue button
8. Now press View .CSV File button and ensure that files have been cleared

• First, check to see if the settings have been changed (size, roundness, sensitivity) and maybe set them back to default. Another user might have changed them. 
• Second, make sure your cells are within the range for the assay (5–60 μm).
• Finally, don‘t just load the cells and proceed; rather, after you put the slide in, let the cells settle for a couple minutes. This will bring most of the cells into the same focal plane prior to imaging.
• You can also use the “Details” button after a count to see how the Countess counted the cells. Live cells with be circled blue, dead cells will be circled red, and objects circled in black are excluded from the count. If cells are not counted or the live and dead discrimination is not accurate, try adjust the settings or focus and recount them.

When you use the test beads, we recommend vortexing the bead stock on high for a full 30 seconds, then pipetting out 10 µL right away to add to 10 µL of trypan blue. The bead and trypan blue mixture should then be pipetted up and down several times to make sure it is well mixed, and the 10 µL sample should be loaded into the slide right away after mixing. The beads can settle quickly, which will affect the concentration. You can also view the beads using the “Details” button to ensure that all beads on the Countess™ screen are counted by the counting algorithm.