Experiments for the determination of the cell division index

Summary

The cytokinesis index is the proportion of dividing cells to all cells in a culture.

Principle

The basic principle of cytokinesis index assay is to detect the proliferative capacity of cultured cells by the index of division. When calculating cell division, it is generally required that the number of cells observed is 1000 or more.

Operation method

Experiments for the determination of the cell division index

Principle

The basic principle of cytokinesis index assay is to detect the proliferative capacity of cultured cells by the index of division. When calculating cell division, it is generally required that the number of cells observed is 1000 or more.

Materials and Instruments

Equipment:
① Cells in adherent wall culture
② 24-well culture plate
③ Pipette
③ Suction tube ④ Gel cap
⑤ Centrifuge tube
⑥ Petri dish
⑦ Coverslip
⑧ Alcohol cotton balls
⑧ Alcohol cotton balls ⑨ Alcohol lamp
⑩ Centrifuge
⑪ Inverted microscope
⑫ General optical microscope
⑫ Ultra-clean bench
Reagents
① D-Hanks liquid
① D-Hanks liquid ② DMEM medium (including 10% calf serum)
③ 0.25% trypsin digestive solution
④ Jimson's stain

Move

The basic procedure for the determination of cell division index can be divided into the following steps:
A. Digest monolayer cultured cells in the logarithmic growth phase with trypsin and collect the cells in a 10 mL centrifuge tube.
B. Centrifuge the cells at 800-1000 r/min for 5 min, and discard the supernatant.
C. Prepare a single-cell suspension by adding DMEM medium (containing 10% calf serum).
D. Blow the cells to homogeneity and count them. E. Inoculate cells at a concentration of 2x104-5x104 cells/mL in coverslips.
E. Inoculate the cells at a concentration of ^2x104-5x104 cells/mL in a culture dish with coverslips.
F. Remove one coverslip every 24 hours and fix it with 95% ethanol for 5 min.
G. Stain the cells on the coverslips with Giemsa's staining solution for 10 min.
H. Identify the dividing cells and nondividing cells by observing the cell structure under the high power objective of microscope. I. Select the cells and identify the dividing cells.
I. Select 3 different areas with high, medium and low cell density, observe 1000 cells in each area, and count and record the number of dividing cells.
J. Calculate the average value of the number of dividing cells in the above 3 areas.
K. Calculate the proportion of dividing cells among 1000 cells, and then obtain the division index of the cultured cells at different time-phase points.
L. Take the time-phase points as the horizontal coordinate, and the division index of the cells at each time-phase point as the vertical coordinate. L. Plot the cell division index of cultured cells at different time-phase points using each time-phase point as the horizontal coordinate and the cell division index at each time-phase point as the vertical coordinate.

Caveat

1. The amount of cells inoculated onto each cover sheet must also be consistent; since dividing cells that are close to or about to complete division can easily be confused with undivided cells, special care must be taken to minimize errors by establishing uniform criteria for division.

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