The importance of accurate cell counting in flow cytometry and cell sorting

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Many flow cytometry and cell sorting protocols recommend a specific cell concentration or range of concentrations for optimal staining and analysis. Incorporating cell counting into the flow cytometry workflow is a simple way to help increase experimental success and potentially save valuable time and money. This procedure enables:

• Confirmation of having a sufficient number of cells for the protocol
• Determination of the optimal amount of staining reagent
• Knowledge of the amount of cells lost during staining
• Verification of staining efficiency

For many flow cytometry staining protocols, the starting cell number or concentration is critical to achieving optimal staining and may dictate the appropriate amount of staining reagents, incubation time, and reaction volume. With an accurate cell count before starting a staining protocol, you can use the appropriate recommended amounts of cells and stains, avoiding waste of valuable reagents and time (Figures 1 and 2).

Applying too little stain can lead to problems such as weak signals, while overstaining can increase background and cause bleed-through of signal into other fluorescence channels, both of which can affect proper discrimination of fluorescent cell populations and waste valuable reagents. Automated cell counters that measure viability can confirm that cells are healthy and alive before you proceed further in a staining protocol.

Having a way to quickly count cells and examine fluorescence after staining is also valuable. Cell loss is inevitable in many staining protocols, so a cell count after staining can determine how many cells were lost and if a concentration step is required before analysis. With over 20 interchangeable light cubes to choose from, the Invitrogen Countess II FL Automated Cell Counter also enables you to quickly visualize the fluorescence of cells to determine the efficiency or quality of cell staining or transduction for a wide variety of dyes and fluorescent proteins, not only GFP and RFP. With other methods such as manual counting, you can determine the total number of cells but not how well the cells are transduced or fluorescently stained; the fluorescence feature of the Countess II FL Automated Cell Counter enables accurate assessment of transduction or staining efficiency (Figure 3), which can diff er even if the total cell number does not change.

The Countess II FL Automated Cell Counter allows you to quickly examine a small sample of cells before analyzing your full cell sample in flow cytometry for a large cell population. Although the Countess II FL Automated Cell Counter is not intended to replace your flow cytometer, as it does not provide data from a large number of events, these analysis methods are complementary to each other. The Countess II FL Automated Cell Counter can be used to quickly screen a representative sample to determine the quality of fluorescence and data trends before more comprehensive flow cytometry analysis (Figure 4). A quick check of cellular fluorescence prior to analysis can confirm that the cells are properly stained or expressing fluorescence as expected before they are analyzed further.

Compared to traditional methods such as manual counting using a hemocytometer and microscope, the Countess II FL Automated Cell Counter provides significant benefits in counting accuracy and time savings. These benefits help to increase experimental confidence and allow more time for flow cytometry acquisition and analysis.

When manually counting cells using a hemocytometer and a microscope, count-to-count variability of a single sample by an experienced technician is typically 10% or more. Counting variability between multiple technicians commonly exceeds 20%. Reliance on operator judgment, regardless of the cell counting method, contributes to this variability. Light intensity and focus settings on a microscope and some automated cell counters are subjective and therefore can contribute to variability. Individual users must also determine which objects to include and exclude in the cell count, as well as decide which cells will be counted as alive or dead. The number of subjective variables in manual counting can thus lead to wide variation in results between users, resulting in inconsistency in the number of cells stained and analyzed in flow cytometry. Automated cell counters minimize the subjective nature of manual counting as well as user-to-user differences in total cell counts (Figure 5).

The additional time it takes to manually count cells (up to 5 minutes) compared to counting with the Countess II FL Automated Cell Counter (10 seconds) is often overlooked as an added cost. An individual counting 5 slides per day (2 samples per slide) can save about 10 hours per month by switching to an automated counter with a reusable slide. The time savings increase to about 15 hours per month when using disposable slides (Figure 6). This additional time may allow cells to be counted at multiple points throughout a flow cytometry workflow without increasing the time it takes to complete an experiment.

Counting cells and measuring viability is quick and easy with the Countess II Automated Cell Counter or Countess II FL Automated Cell Counter— simply stain a 10 μL sample of cells with trypan blue, pipet the stained cells into a chamber slide, then allow the instrument to autofocus and count to obtain the total cell concentration along with the concentration and percentage of live and dead cells (Figure 7). Alternatively, many flow cytometry users today utilize single-channel viability markers, like Invitrogen LIVE/DEAD Fixable Dead Cell Stains, which can also be observed with the Countess II FL Automated Cell Counter (Figure 8). These stains can help users avoid potential fluorescence quenching artifacts due to trypan blue staining.

The ability to easily perform multiple cell counts to determine cell concentration or verify staining efficiency—all before scheduling time on a costly core facility instrument or performing a large-scale experiment—can save time and money. Since most other methods for counting cells are time-consuming or require expensive equipment and calibration, cell counting is often not performed throughout a protocol. The Countess II and II FL Automated Cell Counters offer a quick and simple way to determine cell number and examine cell health and viability. Since total cell counts, viability, apoptosis, and fluorescence can be measured in as little as 10 seconds, these key parameters can easily be confirmed at several points throughout a flow cytometry workflow. The Countess II and II FL Automated Cell Counters can give you the confidence to proceed through your workflow and help ensure a successful experiment.

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Examining cell number, viability, and fluorescence is quick and easy with the Countess II FL Automated Cell Counter.

• Countess II FL Automated Cell Counter (Cat. No. AMQAF1000)
• Countess Cell Counting Chamber Slides (Cat. No. C10228) or Countess II FL Reusable Slide (Cat. No. A25750)
• Invitrogen Trypan Blue Stain (Cat. No. T10282) or fluorescent stain

1. Mix 10 μL of cell sample with 10 μL of trypan blue and pipet 10 μL of the stained sample into a Countess chamber slide.
2. Insert the slide into the sample port of the Countess II FL instrument to initiate autofocus.
3. Press the “Count” button.
4. Optimize results if needed by using the gating options for size, brightness, and circularity. Viewing the histogram while gating can help you determine the appropriate settings for your sample.
5. Record the concentrations and percentages of total, live, and dead cells.
6. Optional: Use the built-in dilution calculator to determine the appropriate cell dilution for your flow cytometry staining protocol.

1. Stain the cell sample (Table 1) according to the flow cytometry staining protocol, or obtain cells expressing fluorescence.
2. Pipet 10 μL of the fluorescent cell sample into a Countess chamber slide.
3. Insert the slide into the sample port of the Countess II FL instrument to initiate autofocus.
4. Adjust light intensities to minimize fluorescence background.
5. Press the “Count” button.
6. Optimize results if needed by using the gating options for size, brightness, circularity, and fluorescence intensity. Viewing the histogram while gating can help you determine the appropriate settings for your sample.
7. Record the concentration and percentage of fluorescent cells.
8. Optional: Use the built-in dilution calculator to determine the appropriate cell dilution for your flow cytometry staining protocol.

Table 1. Dyes and light cubes commonly used with the Countess II FL cell counter for viability and apoptosis determination.