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

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):
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PartA (4Â°C storage)	PartB (-20 Â° 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 (4Â°C storage)	PartB (-20 Â° C preservation)
FITC-Labeled Anti-BrdU mAb	Br-dUTP
PI/Rnase staining buffer	Br-dUTP
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 Â° C for 12-18 hours, and the best results were obtained. Cells can be stored at -20 Â° C

Months. )

2. Prepare the staining solution and use it now.

DNA labeling solution	1 test	5 tests	10 tests
Reaction Buffer	10.00Î¼l	50.00Î¼l	100.00Î¼l
TdT Enzyme	0.75Î¼l	3.75Î¼l	7.50Î¼l
FITC-dUTP	8.00Î¼l	40.00Î¼l	80.00Î¼l
Distilled water	32.2500Î¼l	161.25Î¼l	322.50Î¼l
total capacity	51Î¼l	255Î¼l	510.00Î¼l

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 1Ã—10^6/ml) was added to a centrifuge tube, centrifuged at 300Ã—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 Î¼l of DNA labeling solution.

5) Incubate at 37 Â° 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 Â°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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