Concentration and Viability Determination of Human Leukocytes
Blood contains three main types of cells, red blood cells (erythrocytes), platelets (thrombocytes) and white blood cells (leukocytes). Leukocytes comprise a variety of cell types including granulocytes, monocytes, and lymphocytes. Together, these
cells are essential for both innate and adaptive immune responses, and viability assessments of leukocytes are important in many research investigations.
In this application note, we demonstrate the utility of the Tali® Image-Based Cytometer to count blood cells. When used in conjunction with nucleic acid dyes, the Tali® instrument enables identification of leukoctyes in whole and lysed blood and allows users to obtain quick and accurate leukocyte viability data from lysed blood samples. In addition, each assay run on the Tali® instrument requires only 25 μL of sample, minimizing the amount of blood needed for analysis. Samples are applied to one of the pre-sterilized, individually packaged Tali® Cellular Analysis Slides. The slides are manufactured using low-fluorescence plastic (so they don’t interfere with the instrument readings) and can can be disposed of after use with your regular biohazard waste.
Materials and Methods
Evaluation of whole and lysed blood
Total cell numbers from whole and lysed blood samples were obtained from three platforms: Tali® Image-Based Cytometer, Beckman Coulter® Z1 COULTER COUNTER® Particle Counter, and a hemocytomer. Whole blood from a single donor was collected, and a portion of the sample was diluted to 5 x 106 cells/mL and then split into three tubes. Each tube served as the sample for one of the three counting platforms. Whole blood from the same donor was also lysed using the standard protocol that included an ammonium chloride solution (0.5 M NH4Cl, 10 mM NaHCO3, 1 mM EDTA, pH 7.4). After erythrocyte lysis, the remaining cells were diluted to 1 x 106 cells/mL in Dulbecco’s phosphate-buffered saline (DPBS) and split into three tubes. Again, each tube served as the sample for one of the three counting platforms. A disposable hemocytometer was used (Incyto® C-Chip™ DHC-N01-5), and cells were imaged on a standard upright microscope using a 4x objective and counted manually. Gating on the Tali® instrument and Coulter Counter® Analyzer was set at 3 μM for whole blood and 5 μM for lysed blood. Three technical replicates were performed on each counting platform.
Determination of nucleated cells in whole and lysed blood
To identify nucleated cells, human whole and lysed blood was diluted to approximately 5 x 106 cells/mL. Vybrant® DyeCycle™ Green Stain was used to stain nucleated cells (i.e., leukocytes). Samples were incubated with 2 μL of Vybrant® DyeCycle™ Green Stain per milliliter of blood for at least 30 minutes at 37°C. Analysis was performed using the “GFP Assay” on the Tali® instrument (collection set to 20 fields per slide).
Determination of viability
Human whole blood lysis was performed using the standard protocol (described above). After erythrocyte lysis, the remaining cells were diluted to 5 x 106 cells/mL in DPBS. To determine viability, diluted lysed blood samples were stained using the Tali® Viability Kit - Dead Cell Red according to manufacturer’s instructions. Samples were then divided, and cell viability was determined using both a flow cytometer and the Tali® instrument. On the Tali® instrument, the samples were analyzed using the ‘Viability Assay.’
Figure 1. Quantification of whole and lysed blood. Samples of whole and lysed blood were quantified using the Tali® Image-Based Cytometer, Z1 COULTER COUNTER® counter, and hemocytometer (A). The Tali® Image-Based Cytometer provides images of all the cells counted and visual confirmation of the counting classification (B–E).
Quantification of whole and lysed blood
Quantification of whole and lysed blood was performed on the Tali® Image-Based Cytometer, the Z1 COULTER COUNTER® Particle Counter, and a hemocytomer (Figure 1A). In addition to the cell number and concentration, the Tali® instrument acquires and displays a brightfield image for each field counted. When the ‘circles’ layer is activated in the Tali® instrument software, the image is overlaid with colored circles that indicate counted cells (blue circles) and uncounted particles (black circles) (Figure 1B–E). Using this readout, cell counting accuracy can be viewed and changes in the sensitivity and circularity settings can be made to better fit cells that have varied size and shape.
Detection of DNA-containing cells (leukocyte identification)
Nucleated cells were identified within samples of whole and lysed blood using Vybrant® DyeCycle™ Green Stain. The population of leukocytes in whole blood is small, estimated at less than 1% of the total cell population. However, using the Tali® Image-Based Cytometer, leukocytes stained with Vybrant® DyeCycle™ Green Stain can be easily identified (Figure 2A). After lysis, the blood sample mainly consists of nucleated cells (leukocytes), as evidenced by the abundance of Vybrant® DyeCycle™ Green positive cells (Figure 2B).
Figure 2. A sample of human whole (A) and lysed (B) blood was stained with Vybrant® DyeCycle™ Green Stain and analyzed on the Tali® Image-Based Cytometer. In green, a single leukocyte can be identified among a population of red blood cells (A). After erythrocyte lysis, the population of cells becomes predominately leukocytes, as evidenced by the abundance of Vybrant® DyeCycle™ Green positive cells (B).
The viability of leukocytes in lysed whole blood was measured immediately following the lysis step. Cells were stained with the Tali® Viability Kit - Dead Cell Red reagent. This kit uses propidium iodide (PI) to stain dead cells while viable cells remain unstained. Small cellular debris was gated out of the data analysis, and cell size was determined (Figure 3A). The ‘Cell Size’ histogram displays how many cells of each size are present in the sample. Cells that are PI positive can be identified by setting the threshold based on the fluorescence intensity of unlabeled cells (Figure 3B). Cells with fluorescence intensities to the right of the blue vertical threshold line are counted as positive for PI staining (dead leukocytes). The Tali® instrument also provides visual confirmation in the form of an image for each field of cells counted. Figure 3C is an image from one of these fields. Cells with red fluorescence (PI positive) can be easily identified on this image. Additionally, PI positive and cells are outlined with red circles whereas negative cells are outlined with blue circles. Samples labeled with PI were analyzed on both the Tali® Instrument and a flow cytometer. The percent of cells positive for PI were similar regardless of the analysis platform (Figure 3D).
The Tali® Image-Based Cytometer is an excellent tool for analyzing leukocyte viability. In addition, the Tali® instrument can count cells in whole and lysed blood samples. When cells were stained with Vybrant® DyeCycle™ Green Stain, nucleated cells, such as leukocytes, were easily identified. The Tali® instrument offers an affordable and accurate method to examine blood samples and requires only a small amount of starting sample.