Apoptosis detection: detection method of bio-chemical characteristics.

according to the detection method of the characteristics of

biochemics

1,

agar sugar gel electrophoresis

1) conventional agarose gel electrophoresis

method one:

1. Collect cells (5×106) 1000r/min, centrifuge 5min, go to the top clear.

2. PBS wash once, 1000r / min, centrifuge 5min, go to the qing.

3. Add cell cleavage 500 sl heavy suspended cells, 50 degrees C water bath, 3 to 5h, shake from time to time or 37 degrees C overnight.

4. Add 0.5 ml balanced phenol, upside down several times mix, 6000r/min, centrifugal 5min.

5. Move up to another Ep tube, add 0.5 ml chloroform: isosterol pumping, upside down several times mixed, 6000r/min, centrifugal 5min.

6. Move up to another Ep tube, add 50 μl of 3mol/L sodium acetate and 2 ml of pre-cooled waterless ethanol, upside down several times mixed, visible floc-like white sediment.

7. Liquid nitrogen 5 to 10min, 12 000r/min centrifugation 10min precipitation DNA, go to the upper clear, vacuum dry or blow-dry under the fan residual liquid.

8. Add 50 to 100 μl TE buffer, plus 5 μl RNase, 37 degrees C water bath 30min.

9. Take 20 μl plus sample buffer 2 to 5 μl, 1% agarose gel electrophoresis (voltage 50V, 1.5 to 2h), UV observation.

method two:
1. Centrifugal collection cells (5×106), PBS (no Ca2 plus and Mg2 plus) wash 1 to 2 times.
2. 0.5 ml TBE solution re-suspended, 37 degrees C incubation 30min.
3. 1mg/ml protease 30min at K 37 degrees C.
4. Add 0.1 ml of the sample buffer.
5. 25 sl sample, 1.5% agarose gel electrophoresis, 2V/cm 6h.
method three:
1. Collect cell suspension (106 cells), low-speed centrifugation (1000r/min, centrifugation 5min).
2. To clear, precipitation plus 0.4 ml low seepage buffer action 1h.
3. 13 000r/min, centrifuged 10min, transferred to another Eppendorf tube, plus an equivalent amount of 50% isopropyl alcohol and 0.5mol/L NaCl mixed, liquid nitrogen 5min.
4. 12 000r/min, centrifuged 10min, discarded, precipitated vacuum dry.
5. Add TE 100μl, 65c
heating
10min, take 20μl, plus 1 to 2 sl sample buffer, electrophoresis observation.
method four:
1. Collect cells, 1000r/min centrifuge 5min, go to the qing.
2. Fixed with 70% ethanol pre-cooled ice, it can be stored in a refrigerator at -20 degrees C for a long time.
3. 1000r/min centrifugation 5min, removal of retaining fluid, heavy suspension of cells with about 0.5 ml of PBS.
4. Transfer to the Eppendorf tube, centrifuge with the same method, go to clear.
5. Cells are re-suspended with a 40 μl PC buffer at room temperature of 30 to 60min.
6. 1000r/min centrifugal 5min, suction in the new Eppendorf tube, vacuum concentrate 15min.
7. Add 0.25% NP-40 solution 3 sl, 1mg/ml RNase A solution 3 sl, 37 oC, 30min.
8. Add 3 sl protease K, 37 degrees C, 30min.
9. Add 12 μl sample diluent, containing 1.5% agar sugar electrophoresis of ethyl bromide ingots, observed.