Equipment and reagents: experimental method: Green Salt,White Quartz,Dragon Tooth,Purple Quartz Henan Qiancuntang medicial technology co.ltd. , https://www.qctchineseherb.com
1. Inverted phase contrast microscope with a 10× objective lens;
2. Label the tube with the volume scale (conical bottom);
3. Improved Neubauer blood cell counter;
4. Counter cover glass;
5. A pocket tube or an equal volume plastic container, such as a test tube or centrifuge tube;
6. Pasteur pipettes (short and long);
7. Adjustable volume micro-sampler (100-250μl);
8. Sterile gun tip;
9. Sterile graduated straw (1-10ml), ear wash or automatic pipetting device;
10. 10g/L trypan blue;
11. Hanks balanced salt solution or storage medium;
1. Prepare the counter. Gently wet the sides of the central network area and use the pressure of the two thumbs to slide the coverslip horizontally from the edges. When the Newton ring (rainbow) is visible on either side of the central marking zone, the coverslip is in the correct position;
2. Centrifuge the cell suspension at 1000 r/min for 5 min and discard the supernatant. Resuspend the cells in a known volume of medium;
3. Blow the cell suspension back and forth several times with a Pasteur pipette and mix to determine that the cells are evenly dispersed;
4. Pipette 250 μl of the mixed cell suspension into a pocket tube using a micropipette. An equal volume of trypan blue solution was pipetted into the cell suspension using a clean applicator tip. Use your thumb and forefinger to quickly flick the pocket tube several times to mix it. Immediately pipette a drop of cell suspension with a Pasteur pipette tip and gently pour it onto the edge of the coverslip near the counting area. Capillary action moves the cell suspension under the coverslip and fills the area between the central and marginal grooves. Repeat this operation on the other side of the coverslip;
5. Place the counter on the microscope stage and move the stage until the 25 small square grids defined by the three parallel lines are centered. The total number of cells (stained and unstained) in 25 small squares (each of which is subdivided into 16 smaller squares for counting). In order to avoid repeating the same cell count, only the three lines of the upper line, the left hand line and the central area of ​​the cells are separated. If the number of cells is large, a counter can be used;
6. When counting the total number of cells, repeat the count in the same area and count only the blue-stained (dead) cells. Calculate the average number of dead cells, and subtract the number of dead cells from the total number of cells to obtain the number of living cells;
7. Calculate the survival rate of the cell population using the following formula:
Survival rate = number of viable cells / (number of viable cells + number of dead cells) × 100%
8. Calculate the cell concentration of the cell suspension as follows:
Average cell number of 1 large cell (25 cells) × 10 4 = cell number / ml
9. Total cell count of cell suspension = cell number / ml × total volume
10. Total viable cell count of cell suspension = total cell number × survival rate (%)
Cell counting and viability assay
Cell count and viability assay