Electronic cell counting by means of a resistor
Electronic cell counting by means of a resistor
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.
Operation method
Title: Scheme 21.2 Electron cell counting experiment by means of a resistor
Principle
The cell samples were diluted in electrolyte (saline or D-PBSA ) and transferred to the lower part of the perforated tube, allowing 0.5 ml of the sample solution to flow through the counter to count the number of cells in it. Move makings For more product details, please visit Aladdin Scientific website.
Sterile: cell culture, D-PBSA, 0. 2 5 % crude trypsin, growth medium
Non-sterile: counting cups
Procedure
1. Digest cells in monolayer culture with trypsin or collect samples from suspension culture. Mix the cell suspension well so that it is as well dispersed as possible into individual cells.
2. In a 25 ml beaker or one of the sample cups, dilute the cell suspension with counting liquid at 1 : 50 to 20 ml. The use of an automated disperser (Fig. 5.20 ) will speed up the dilution and will result in better reproducibility.
Caution Rapid dispensing of the counting solution will create air bubbles that will be counted as cells as they pass through the wells. Therefore, the counting solution should be allowed to stand for a while before counting begins. If the solution is dispensed first and then the cells are added, the likelihood of bubbles is minimal.
3 . Mix the cell suspension well and transfer it to the bottom of the perforated tube, making sure that it covers the small wells and that the external electrode in the sample cup is below the level of the counting solution.
4 . Check the program settings:
(a) Threshold setting (the lower limit of cell size is usually 7. 0um ).
(b) Assay volume (usually 0.5 ml);
(c) Background removal (as required);
(d) Dissolution setting (e.g., 50 if 0.4 ml of D-PBSA in 20 ml is counted).
5. Check the visible analog display
(a) Ensure that all cells are within the set critical range;
(b) Check for cell viability or cell fragmentation (indicated by the shoulder of the curve or by a histogram below the lower limit of the normal threshold).
(c) Check for aggregation of cells into clusters (i.e., particles appearing above the normal size range).
6 . Initiate the counting program.
7 . When the counting cycle is complete, the distribution of cell sizes will appear on the analog display (Fig. 21.4). The number of cells per milliliter can be obtained by converting to the digital display.
