1.Take 20 µl of blood sample and dilute the sample in 180 µl of PBS.
2.Add 12µl AO/PI solution into 12µl sample, gently mixed with pipette;
3.Draw 20µl mixture into chamber slide;
4.Allow the cells to settle in the chamber for around 1 minute;
5.Insect the slide into Countstar FL instrument;
6.Choose the “AO/PI Viability” assay, then Enter Sample ID for this sample.
7.Select Dilution ratio, Cell Type, the click ‘Run’ to start the test.
Step 6: Enter Sample ID for this sample
Step 7: Select Dilution and Cell Type
Caution: AO and PI is a potential carcinogen. It is recommended that the operator wear personal protective equipment (PPE) to avoid directly contact with skin and eyes.
1. Bright Field Image of whole blood
In the bright field image of the whole blood, WBC’s are not visible among the red blood cell. (Figure 1)
Figure 1 Bright field image of whole blood.
2. Fluorescence Image of whole blood
The AO and PI dye are both stains DNA in the cell nucleus of cells. Therefore, Platelets, red blood cells, or cellular debris are unable to affect leukocytes concentration and viability result. Live leukocytes (Green) and dead leukocytes (Red) are easily visualized in the fluorescence images. (Figure 2)
Figure 2 Fluorescence Images of whole blood. (A). Image of AO Channel; (B) Image of PI Channel; (C) Merge images of AO and PI Channel.
3. Concentration and viability of leukocytes
The Countstar FL software automatically count cells of three chamber sections and calculates average value of total WBC cell count (1202), concentration (1.83 x 106 cells/ml), and % viability (82.04%). The whole blood images and data can be easily exported as PDF, Image or Excel for additional analysis or data archiving.
Figure 3 Screenshot of Countstar Rigel Software