Cell cycle detection service

Experiments are divided into four main steps:

1. Cell laying

HeLa S3 cells, density is 80% left and right, make the spread density at 50 % Or so, in order to meet the requirements of flow cytometry, the number of cells is greater than 1 x 10 groups. 2. Cell synchronization treatment:

a) THYMIDINE THYMIDINE was added 18h u200b u200b

b) washed 4 times with incubation PBS, replace fresh medium, cultured 9h [123 ]

c) Add 2 mm thymidine again to synchronize 18h after 18h

3. Sample collection;

a) charge release 2H (S), 6H (G2 / M) And 12H (G1) Cell sample

b) Each sample is charged 4 times, 1000g centrifuge for 5 min, discarded

c) 100 ul PBS resuscitation The cell suspension was sued in the pipette, and 900 u0026 mu; L pre-cooled 70% ethanol was added to the tube of the cell, and the vortex oscillator was dropped into the cell suspension.

D) 4C fixed one hour or longer.

E) 1500RMP, centrifugation 10 min, and remove the fixture.

f) Cell dyed liquid: 50XRNase A. Mother liquor: 50 xpi mother liquor: PBS u003d 20: 20: 960; RNase A mother liquid concentration 1 mg / ml, working concentration 20 u0026 mu; g / ml; pi mother liquor concentration 2.5mg / ML, working concentration 50 u0026 mu; g / ml.

g) Cell 37 degrees dyed for 1 h. According to the cell boy, a volume of cell dye (1 to 1.5 mL) is added, and the cell pass rate is

200 ~ 350Cl / s, and the dyeing liquid is not too much or too small. Excessive, the cell density is too low, and the cell bond is caused by too little.

H) 300 mesh screen is filtered into the flow tube.

4. Upper machine detection;

Flow cytometry is detected on the machine, counting 50,000 cells.

5. Synchronized Data Analysis

Data Using Modfit Software Analysis.