WHITE PAPER Countess II and II FL Automated Cell Counters Increasing cell concentration with constant dye concentration 700 900 1,500 450 The importance of accurate cell 1,500counting 800 600 400 1,500 700 350 500 1,000 600 300 1,000 400 in flow cytometry and 1,000cell sorting 500 250 400 Count 300 Count Count Count Count Count 200 500 500 300 150 200 500 200 100 100 100 50 0 0 0 0 0 0 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Low cell concentration, poor results Optimal cell concentration High cell concentration, poor results Introduction A 16,000 cells B 1,800 cells Many flow cytometry and cell sorting protocols recommend 1,250 125 a specific cell concentration or range of concentrations for 1,000 100 optimal staining and analysis. Incorporating cell counting 750 75 Count Count into the flow cytometry workflow is a simple way to help 500 50 increase experimental success and potentially save 250 25 valuable time and money. This procedure enables: 0 0 50 100 150 200 250 50 100 150 200 250 • Confirmation of having a sufficient number of cells for Fluorescence (x 1,000) Fluorescence (x 1,000) Typical histogram appearance, Ragged histogram appearance, the protocol useful statistics questionable statistics • Determination of the optimal amount of staining reagent Figure 1. Having a sufficient number of cells is required for optimal cell cycle analysis with flow cytometry. Live Jurkat cells were labeled with Invitrogen™ Vybrant™ DyeCycle™ Orange Stain, which is a cell- • Knowledge of the amount of cells lost during staining permeant dye that fluoresces upon binding to DNA. Different numbers of cells were then acquired for cell cycle analysis. (A) Acquisition of 16,000 • Verification of staining efficiency cells produced a cell cycle histogram with a typical appearance and useful statistics, while (B) acquisition of only 1,800 cells produced a cell cycle histogram with a ragged appearance and questionable statistics. Are you sure you have the optimal concentration of viable cells prior to staining? What is the appropriate amount of stain needed for For many flow cytometry staining protocols, the starting the sample? cell number or concentration is critical to achieving optimal Applying too little stain can lead to problems such as weak staining and may dictate the appropriate amount of staining signals, while overstaining can increase background and reagents, incubation time, and reaction volume. With an cause bleed-through of signal into other fluorescence accurate cell count before starting a staining protocol, you channels, both of which can affect proper discrimination of can use the appropriate recommended amounts of cells fluorescent cell populations and waste valuable reagents. and stains, avoiding waste of valuable reagents and time Automated cell counters that measure viability can confirm (Figures 1 and 2). that cells are healthy and alive before you proceed further in a staining protocol. Increasing cell concentration with constant dye concentration 700 900 1,500 450 1,500 800 600 400 1,500 700 350 500 1,000 600 300 1,000 400 1,000 500 250 400 Count 300 Count Count Count Count Count 200 500 500 300 150 200 500 200 100 100 100 50 0 0 0 0 0 0 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Fluorescence (x 1,000) Low cell concentration, poor results Optimal cell concentration High cell concentration, poor results 16,000 cells 1,800 cells Figure 2. Effect of cell concentration on cell cycle1,250 analysis by flow cytometry. Different concentrations of live Jurkat cells were labeled with a 125 constant concentration (10 µM) of Vybrant DyeCycle Orange1,000 Stain. Using the same concentration of stain produced poor cell cycle histograms with both low and high cell concentrations, while staining with the optimal cell concentration100 of 1 x 106 cells/mL gave the proper cell cycle histogram. 750 75 Count Count 500 50 Did your staining achieve the expected250 results? The25 Countess II FL Automated Cell Counter allows you to 0 0 Having a way to quickly count cells and examine50 100 150 200 250 quickly50 100examine150 200 250 a small sample of cells before analyzing Fluorescence (x 1,000) Fluorescence (x 1,000) fluorescence after staining is also valuable. CellTypical loss histogram isappearance, your Raggedfull histogram cell appearance, sample in flow cytometry for a large cell useful statistics questionable statistics inevitable in many staining protocols, so a cell count after population. Although the Countess II FL Automated Cell staining can determine how many cells were lost and if a Counter is not intended to replace your flow cytometer, as concentration step is required before analysis. With over 20 it does not provide data from a large number of events, interchangeable light cubes to choose from, the Invitrogen™ these analysis methods are complementary to each other. Countess™ II FL Automated Cell Counter also enables you The Countess II FL Automated Cell Counter can be used to quickly visualize the fluorescence of cells to determine to quickly screen a representative sample to determine the efficiency or quality of cell staining or transduction for a the quality of fluorescence and data trends before more wide variety of dyes and fluorescent proteins, not only GFP comprehensive flow cytometry analysis (Figure 4). A and RFP. With other methods such as manual counting, quick check of cellular fluorescence prior to analysis can you can determine the total number of cells but not how confirm that the cells are properly stained or expressing well the cells are transduced or fluorescently stained; the fluorescence as expected before they are analyzed further. fluorescence feature of the Countess II FL Automated Cell Counter enables accurate assessment of transduction or A 500 staining efficiency (Figure 3), which can differ even if the total cell number does not change. 49.50% 250 Count 0 100 101 102 103 104 105 106 GFP (BL1-H) B Figure 3. Gating based on fluorescence intensity. Two fluorescence colors are present, each of which can be gated by size, brightness, circularity, and fluorescence intensity. The dim cells (around 50 RFU) have Figure 4. Fluorescent cell counts similar to those obtained by flow been excluded from the GFP-positive count, as indicated by the light- cytometry can be easily acquired with the Countess II FL Automated colored portion of the bars. Cell Counter, with a single light cube. U2OS cells were transduced using Invitrogen™ CellLight™ Nucleus-GFP, BacMam 2.0, and allowed to incubate for 36 hours. The cells were evaluated for GFP on (A) a flow cytometer and (B) the Countess II FL Automated Cell Counter. The proportions of transduced cells determined by the two methods are nearly identical. Manual vs. automated counting Time savings Compared to traditional methods such as manual counting The additional time it takes to manually count cells (up to using a hemocytometer and microscope, the Countess II 5 minutes) compared to counting with the Countess II FL FL Automated Cell Counter provides significant benefits in Automated Cell Counter (10 seconds) is often overlooked counting accuracy and time savings. These benefits help to as an added cost. An individual counting 5 slides per day increase experimental confidence and allow more time for (2 samples per slide) can save about 10 hours per month flow cytometry acquisition and analysis. by switching to an automated counter with a reusable slide. The time savings increase to about 15 hours per month Reduced user variability for increased when using disposable slides (Figure 6). This additional counting accuracy time may allow cells to be counted at multiple points When manually counting cells using a hemocytometer throughout a flow cytometry workflow without increasing and a microscope, count-to-count variability of a single the time it takes to complete an experiment. sample by an experienced technician is typically 10% or more. Counting variability between multiple technicians 80 74 commonly exceeds 20%. Reliance on operator judgment, Switch to cell counter with reusable slide 70 Switch to cell counter with disposable slide regardless of the cell counting method, contributes 60 to this variability. Light intensity and focus settings on 48 50 a microscope and some automated cell counters are 40 subjective and therefore can contribute to variability. 30 30 Individual users must also determine which objects to 19 20 15 include and exclude in the cell count, as well as decide 10 Time saved (hr/month) which cells will be counted as alive or dead. The number 10 of subjective variables in manual counting can thus lead 0 to wide variation in results between users, resulting in 5 slides 10 slides 25 slides inconsistency in the number of cells stained and analyzed Average number of slides counted per day in flow cytometry. Automated cell counters minimize the Figure 6. Estimated hours per month saved by switching from manual subjective nature of manual counting as well as user-to- cell counting to an automated cell counter. user differences in total cell counts (Figure 5). Options for automated cell counting Counting cells and measuring viability is quick and easy Countess II instrument with the Countess II or II FL Automated Cell Counter— 30 Hemocytometer simply stain a 10 µL sample of cells with trypan blue, 25 pipet the stained cells into a chamber slide, then allow the instrument to autofocus and count to obtain the total cell 20 concentration along with the concentration and percentage 15 of live and dead cells (Figure 7).