Method for determining cell growth status

The growth state of cells is closely related to the progress of experiments, and accurate determination of cell state is crucial. The determination of cell state mainly include the following three methods:

1. Morphological observation (simplest and most intuitive)

Cells that have grown well after passage or fluid change are observed under a microscope, with large transparency, strong refractive and unclear contours. The fine structure of some cells can be seen clearly by phase contrast microscopy, and many cells in the dividing stage can also be seen when the cells are in the logarithmic growth stage. When the cell growth state is compromised, the refractive nature of the cells becomes weaker, the contour is enhanced; vacuoles, lipid droplets, and granular matter often appear in the cytoplasm; the gaps between cells increase, and the cells become irregular and lose the characteristics of normal growth.

2. Biochemical testing (molecular level measurement)

Principle: Detection is carried out using biochemical markers unique to cells in different states.

Common methods:

1. Trypan blue/acridine orange staining: The cell membrane of dead cells is incomplete, and the dye will enter the cell to turn it blue, while the living cells will refuse to be stained. This is the most classic method of counting cell viability.

2. Fluorescence staining/flow cytometry:

   1) Annexin V/PI double staining: It is the "gold standard" for distinguishing between early apoptosis (Annexin V+), late apoptosis/necrosis (Annexin V+/PI+) and live cells (double negative).

   2) DAPI/Hoechst: Used to observe the morphology of the nucleus, the apoptotic nucleus will appear in the form of dense and densely stained fragments.

3. Detect caspase enzyme activity: Caspase is a key executor in the apoptosis process, and its increased activity is a clear signal of apoptosis.

4. Functional testing (to examine the "working ability" of cells).

1. MTT/CCK-8 assay: Detect the metabolic activity of cells and indirectly reflect cell proliferation and viability. The higher the activity, the greater the absorbance value.

2. Clone formation experiments: Evaluate the proliferation and viability of individual cells, and cells that can form large clones are the real "strong".

Determining cell state is a comprehensive art, which often requires the combination of multiple technologies, from morphology, biochemistry to function, to cross-validation to draw the most accurate conclusions.