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title How to test apoptosis

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Provider: Shanghai huding Biotechnology Co., Ltd Release time: July 30, 2020 reading times: 38 times >>Enter the company's stand
1、 PI single staining
1. The number of cells {about (1-5) × 106 cells / ml} was collected, centrifuged at 500-1000 R / min for 5 min, and the culture medium was discarded.
2. 3 ml PBS washing once.
3. Centrifugation to remove PBS, adding ice precooled 70% ethanol to fix, 4 ℃, 1-2 hours.
4. Centrifugation was used to discard the fixative, and 3ml PBS was suspended for 5min.
5. Filter once with 400 mesh screen, centrifuge at 500-1000r / min for 5min, and discard PBS.
6. Stain with 1 ml PI dye solution and keep away from light at 4 ℃ for 30 min.
7. Flow cytometry detection: Pi fluorescence was excited by argon ion. The laser wavelength was 488nm, and the emission wavelength was more than 630nm. Red fluorescence was generated. The histogram of PI fluorescence intensity was analyzed, and the scatter diagram of the opposite side of the forward scattered light could be analyzed.
8. Results: compared with normal cells, the forward scattered light of apoptotic cells decreased, while the side scattered light could be high or low, which was related to the type of cells. When analyzing the histogram of PI fluorescence, the double or aggregated cells and the cell fragments with weak fluorescence were excluded by gate technology On the fluorescence histogram, apoptotic cells appeared a sub diploid peak before G1 / G0 phase. If the fluorescence intensity of G1 / G0 phase is 1.0, the fluorescence intensity of sub diploid peak of a typical apoptotic cell sample is 0.45. PI fluorescence intensity of chicken and salmon red blood cells is 0.35 and 0.7 respectively, which can ensure that there is no cell fragment but complete cell between them.

matters needing attention:
The decrease of DNA dyeability is considered to be one of the markers of apoptotic cells. However, the decrease of DNA dyeability may be due to the decrease of DNA content or the change of DNA structure which changes its ability to combine with dyes. Care should be taken when analyzing the results.

2、 Annexin V / PI double staining

1. Cell collection: the suspension cells were directly collected into a 10 ml centrifuge tube, the number of cells per sample was (1 ~ 5) × 106, / ml, 500 ~ 1000r / min, centrifuged for 5min, and the culture medium was discarded.
2. Wash once with incubation buffer and centrifugate for 5 min at 500-1000 R / min.
3. Cells were resuspended with 100ul labeled solution and incubated in dark for 10-15min at room temperature.
4. Centrifugation at 500-1000 R / min for 5 min precipitated cells, incubated with buffer solution and washed once.
5. Add the fluorescence (sa-flow) solution and incubate at 4 ℃ for 20min, avoid light and vibrate from time to time.
6. Flow cytometry analysis: the excitation wavelength of flow cytometry was 488 nm, FITC fluorescence was detected by a 515 nm passband filter, and PI was detected by a filter with a wavelength greater than 560 nm.
7. Results: apoptotic cells were resistant to all dyes such as pi, but necrotic cells could not.

Conclusion: the DNA of cells with damaged cell membrane can be stained with PI to produce red fluorescence, while cells with intact cell membrane will not produce red fluorescence. Therefore, PI was not stained in the early stage of apoptosis without red fluorescence signal. Normal living cells are similar to this. On the scatter plot of bivariate flow cytometry, the lower left quadrant showed living cells (FITC - / pi -); the upper right quadrant showed non living cells, i.e., necrotic cells (FITC + / PI +); and the lower right quadrant showed apoptotic cells (FITC + / pi -)

3、 Heochst 33342 / PI double staining method

1. The suspension growth cells were added with heochst 33342 at a final concentration of 1 ug / ml and incubated at 37 ℃ for 7-10 min.
2. Centrifugation at 500 ~ 1000r / min for 5min and discard the dye solution.
3. Add 1.0ml PI dye solution and dye for 15min at 4 ℃.
4. Filter once with 400 mesh screen.
5. Flow cytometry analysis: the ultraviolet fluorescence of heochst 33342 was excited by krypton laser, the excitation wavelength was 352nm, the emission wavelength was 400 ~ 500nm, blue fluorescence was produced; PI was excited by argon ion laser, the excitation wavelength was 488nm, the emission wavelength was more than 630nm, and red fluorescence was produced. The scatter map or topographic map of blue fluorescence versus red fluorescence was analyzed.
6. Result judgment: on the scatter diagram of blue fluorescence to red fluorescence, the results showed that normal cells were low blue light / low red light, apoptotic cells were high blue light / low red light, and necrotic cells were low blue light / high red light.

matters needing attention:
① The migration of apoptotic region to necrotic region was also observed on the scatter diagram of red fluorescence to blue fluorescence, which may be due to the further degradation of DNA of apoptotic cells.
② The incubation time of cells with heochst 33342 dye should not be too long, and it should be controlled within 20 minutes. If it is too long, the emission spectrum of heochst 33342 will shift from blue to red, and the ratio of red fluorescence to blue fluorescence will change, which will affect the judgment of the results.


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