Digest the monolayer culture with trypsin, or take a sample from the suspension culture; prepare a coverslip and add the cells to the small chamber of the hemocyte counting plate. Count the cells under a microscope and calculate the cell concentration. Source: Animal Cell Culture: a Guide to Basic Techniques, Fifth Edition
Operation method
Scheme 21.1 Cell counting experiments with blood counting plates
Principle
Digest the monolayer culture with trypsin, or take a sample from the suspension culture; prepare a coverslip and add the cells to the small chamber of the hemocyte counting plate. Count the cells under a microscope and calculate the cell concentration. Move makings For more product details, please visit Aladdin Scientific website.
Sterile: D-PBSA, 0.25% crude trypsin, growth medium, pipette yellow tips
Non-sterile: pipette, 20ul or 100ul adjustable, hemocyte counting plate (modified Neubauer), digital counter, microscope
Procedure
1. Cell sampling
(a) Monolayer culture
(i) Trypsinize cells from monolayer cultures as for conventional cultures (see Protocol 13.2), and resuspend the cells in medium to a concentration of approximately 1X106 cells/ml. samples should be counted without removing the tryptic digest, but should be blown up and dispersed with the tryptic solution, and then counted. If the cells are clumped, dilute them doubly (50:50) with serum-containing medium and count again.
(ii) Mix the cell suspension well so that it is as well dispersed as possible into individual cells, and transfer a small portion of the sample (about 1 ml) to a vial or common container.
(b) Suspension culture
(i) Mix the cell suspension well so that the cells are dispersed as much as possible without aggregation.
(ii) Transfer 1 ml of cell suspension to a vial or common container.
In this method, the minimum concentration of cells required is roughly 1X106 cells/ml, so the cell suspension needs to be centrifuged (100 g, 2 min) and resuspended in a smaller volume for easy counting.
2. Preparation of the Hematocrit Plate
(a) Clean the surface of the plate with 70% ethanol, taking care not to scratch the silver surface.
(b) Wash the coverslip, dabbing the edges slightly with water, and press the coverslip onto the grooves of the counting plate and onto the half-silver counting area (Fig. 21.1). When an interference image appears ('Newton's ring', or a colorful rainbow ring between the counting plate and the coverslip similar to an oil droplet on water), it means that the coverslip has been placed correctly and the depth of the counting chamber has been determined.
3. Mix the cell samples thoroughly, blow vigorously to disperse all cell clumps, and then aspirate a 20?l sample with a Pasteur pipette or pipette tip.
4. Immediately move the cell suspension to the edge of the hemocyte counting plate chamber and squeeze the cell suspension from the pipette, using capillary action to fill the void between the counting plate and coverslip. There should be neither too much nor too little liquid in the chamber; the liquid should flow just to the edge of the counting plate groove, otherwise the volume of the chamber will change due to the change in surface tension.
5. Fill the other chamber of the counting plate with cell suspension as above.
6. Blot up the excess liquid (but do not bring out the cell suspension under the coverslip) and place the counting plate on the microscope carrier.
7. Select a 10X objective and focus using the gridlines (Fig. 21.1). If focusing is difficult due to weak contrast, the variable diaphragm can be adjusted to reduce the amount of incoming light, or the light can be slightly tilted by biasing the concentrator.
8. Move the counting plate so that the field of view under the lens is exactly one large square in the center of the entire network area and is as far as you can see bounded by 3 parallel lines. Because of the eyepiece, it is possible that the four corners of the large square may lie slightly outside the field of view (Figure 21.1).
9. Count the number of cells within this 1 mm2 area using finer delineated areas (again delineated by 3 parallel lines) and a single grid line. To avoid double counting, only the left and upper lines are counted for cells in the pressure line; the right and lower lines are not counted. For routine subculture, a cell count of 100~300 cells/mm2 is sufficient; the more cells counted, the more accurate the result will be. The more cells counted, the more accurate the calculation results. For experiments that require precise quantification, the number of cells counted should be 500~1000.
(a) If there are too few cells in the field of view ( <100/mm2 ), one or more large squares (1 mm2 each) around the center large square need to be counted additionally.
(b) If there are too many cells in the field of view ( >100/mm2 ), count only the 5 small squares (each separated by 3 parallel lines) on the diagonal of the large square (1 mm2 ).
10. If the counting plate has two chambers, the second chamber can be moved into the field of view for a second count. Alternatively, the plate can be rinsed and the cell sample can be added for a second count.
