Experimental methods for cell counting and survival testing.

Biotechnology Channel News: Cell freezing and resuscitation is the routine work of cell culture, which can solve the degeneration or transformation of cells due to continuous generation.
cell counts are needed for primary and lateral culture of cells, as well as for cell freezing and resuscitation.
are the test methods for cell counting and survival? 1, principle: (1) calculate the number of cells can be used blood cell count disk or Coultercounter particle counter automatic count.
(2) the blood cell count disk generally has two chambers, each chamber is engraved with 9 1mm2 squares, of which 4 corners of the square and then finely engraved 16 squares, the depth is 0.1mm.
when the cover slide is covered above the chamber, the volume of each large square is 1mm2 x 0.1mm x 1.0x10-4ml.
use, count the number of cells in each square, multiply by the dilution multiplier, and multiply by 104, i.e. the number of cells per ml.
(3) the survival test step is dyexclusion, which uses dyes to penetrate dead cells and color them, while living cells do not color because the cell membrane is intact and the dyes cannot penetrate.
use blue trypan blue dyes in general, and red Erythrosin blue if cells are not easy to absorb.
cell survival ratio is calculated: number of living cells / (number of living cells plus number of dead cells) x 100%.
count should be completed within minutes of Tai Panlan dyeing, over time, some living cells also began to ingest dyes, because Tai Panlan has a strong affinity for protein, with serum-free dilution, can make dyeing count more accurate.
2, Materials: 0.4%w/v trypan blue (GibcoBRL15250-061); Erythosin bluish stain; 0.1gram Erythrosin bluish (Sigma E-9259) and 0.05gram preservative methyl para SigmaH-3647 is dissolved in 100ml Ca/Mg-freesaline; hemocytometerandcoverslips; counters; low-fold inverted microscopes; particle counters (Coultercounter, CoulterElectrons).
white blood cell dilution (4% acetic acid solution).
3, step: (1) Take 50 sl cell suspension and 50 sl trypan blue (or Erethrosinbluish) and other volume mix evenly in 1.5 ml small centrifellular tube.
(2) Take a little mixture (about 15 sl) from the groove above the blood cell count platechamber, cover the cover slide, observe under a 100-fold inverted microscope, live cells do not stain, and dead cells are blue (or red-Erythrosinish blush).
(3) counts the total number of cells in the four squares, divided by 4, multiplied by the dilution multiplier (at least multiplied by 2, due to mixing with volumes such as trypanblue), and finally multiplied by 104, i.e. the number of cells per ml of cell suspension.
if the cell is on the line, only the cells on the upper and right lines (or the cells on the lower and left lines) are counted.
Note: The total number of 4 large cells x dilution multiplier x 104/4 x number of cells / ml;
high concentration of cell suspension, can be removed for dilution or continuous dilution after counting.
5, example: T75 monolayer culture made 10 ml cell suspension, 0.1 ml solution and 0.1 ml trypan blue mixed evenly in the test tube, take a little mixture added to the blood cell count disk, count the number of cells in the four squares.
live cells/squares: 55, 62, 49, 59; Number of dead cells/squares: 5, 3, 4,6; total number of cells: 243 Average cells/squares: 60.75; Dilution multiplier: 2; Number of cells/ml: 60.75 x 104 x 2 (dilution multiplier) x 1.22 x 106; cell count/flask (10ml): 1.22 x 106 x 10ml x 12.2 x 106; survival rate: 225/243 x 92.6%.
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