Apoptosis detection protocol 2--BrdU detection of apoptotic cell DNA fragmentation

July 29 21:08:42, 2023

Apoptosis detection operation flow
2. BrdU detects DNA fragment fragments of apoptotic cells
Related reagents and components:


One-step method: APO-DIRECT kit (Cat. No. 556381):
 

PartA (4°C storage) PartB (-20 ° C preservation)
PI/RNase A Solution FITC-dUTP
Reaction Buffer TdT Enzyme
Rinsing Buffer Negative Control Cells: Fixed cells
Wash Buffer Positive Control Cells: Fixed cells

Two-step method: APO-BRDUTM kit (Cat. No. 556405):
PartA (4°C storage) PartB (-20 ° C preservation)
FITC-Labeled Anti-BrdU mAb Br-dUTP
PI/Rnase staining buffer
Reaction Buffer TdT Enzyme
Rinsing Buffer Negative Control Cells: Fixed cells
Wash Buffer Positive Control Cells: Fixed cells
Experimental procedure (for APO-DIRECT kit):

1. Cell fixation:
1) After washing the cells with PBS, the cells were resuspended in 1% paraformaldehyde in PBS at a concentration of 1-2 x 106 / ml and ice bath for 30-60 minutes.
2) Centrifuge at 300 xg for 5 minutes and discard the supernatant.
3) Wash the cells twice with 5 ml PBS, resuspend in 70% ethanol at a concentration of 1-2 x 106 / ml, and ice bath for 30-60 minutes. (70% ethanol
The cell suspension was tested for apoptosis after being placed at -20 ° C for 12-18 hours, and the best results were obtained. Cells can be stored at -20 ° C
Months. )
2. Prepare the staining solution and use it now.
DNA labeling solution 1 test 5 tests 10 tests
Reaction Buffer 10.00μl 50.00μl 100.00μl
TdT Enzyme 0.75μl 3.75μl 7.50μl
FITC-dUTP 8.00μl 40.00μl 80.00μl
Distilled water 32.2500μl 161.25μl 322.50μl
total capacity 51μl 255μl 510.00μl


3. Cell staining:

1) Take the centrifuge tube and the Falcon tube and number them in the order of the specimen.

2) Add 1x106 cell suspension to the centrifuge tube, centrifuge at 300xg for 5 minutes, discard the ethanol, leaving the deposited cells.

Negative Control Cells (Positive Control Cells): After mixing, 1 ml (cell number about 1×10^6/ml) was added to a centrifuge tube, centrifuged at 300×g for 5 minutes, and ethanol was discarded to leave deposited cells.

3) Add 1.0ml Wash Buffer to each tube, wash the cells twice, and discard the supernatant.

4) Resuspend the cells by adding 50 μl of DNA labeling solution.

5) Incubate at 37 ° C for 60 minutes (or overnight at room temperature) and shake once every 15 minutes. (The incubation time of non-quality control cells at 37 °C needs to be neglected according to different conditions.)

6) Add 1.0 ml of Rinse Buffer to each tube, centrifuge at 300 xg for 5 minutes, and discard the supernatant. Repeat twice and discard the supernatant.

7) Add 0.5ml PI/RNase A Solution and mix. If the cell concentration is low, adjust the dosage to 0.3 ml.

8) Incubate at room temperature for 30 minutes in the dark.

9) Results of up-flow cytometry within 3 hours.


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