JRD357 apoptosis experiment

Cell apoptosis Principle:

This experiment was 1 u0026 MU; g / ml tritokine-based HT in vitro induced cultured HL-60 cells, and there were also a few cells in necrosis. Cells were dual stapled with Hoechst33342 and POIDIUM IODIDE (PI) to distinguish apoptosis, necrotizing and normal cells. Tribese (HT) is an anti-tumor drug that has a good efficacy of acute granulocyte leukemia and acute monocyte leukemia in my country. Studies have shown that HT is in 0.02 to 5 u0026 Mu; G / mL is used for 2 hours, which can induce apoptosis of HL-60 cells, and exhibit a typical apoptosis.

Cell film is a selective biofilm, and the general biofanmeters such as PI cannot pass through the plasma film. When cell necrosis, the plasma membrane is incomplete, and Pi enters the interior of the cell, which can be embedded in DNA or RNA, making necrotic cell coloring, apoptotic cells and live cells. Some live cell dyes can be entered into live cells across membranes due to lipophilic substances, and thus live cell staining can be carried out. Hoechst33342 is an active fluorescent dye and is weak toxic, which is a derivative of bisphenylmidazole. A apoptotic cells and living cells are shown to DNA specific binding (mainly in a A-T) base region. Those who see apoptotic body are apoptotic cells.


Material Human early grain leukemia HL-60 cell reagent, kit
three tips Pine ester base Tris-HCLEDTA buffer alkaline lysate SDS sodium acetate lycetanol ethanol bromide blue sucrose indicator TBE electrophoresis buffer agarose bromide ethyl ingot PI mother liquor HO33342 mother liquor instrument, supplies fluorescence microscope electrophoresis Electric swim tank micro-samples Experimental step 1, reagent preparation

1. u0026 Nbsp; Tripropine (HT): 300 u0026 mu; g / ml;

2. Tris-HCl (pH 7.5): 100 mmol / L;

3. EDTA buffer: 5mol / L;
4. Alkaline Cracking: 0.2 mol / l NaOH;

5. u0026 Nbsp; Sodium acetate: 3mol / l Kac (pH 4.8);

6. pi mother liquor: 500 u0026 mu ; g / ml;

7. HO33342 mother liquor: 2mmol / L;

8. u0026 Nbsp; Human early grain leukemia HL-60 cells: RPMI1640 medium containing 10% calf serum at 37 ° C , Cultured under 5% CO2 conditions.
9. u0026 Nbsp; Other: Isopropanol, 1% SDS, 70% ethanol, bromophenol blue, sucrose indicator, TBE electrophoretic buffer, 1% agarose, bromo ingot.

II, tritokine induced HL-60 cell apoptosis

1. u0026 Nbsp; about 24 hours before the experiment, inoculate two bottles of HL- 60 cells, labels 1, 2, each bottle of about 6 ml of culture solution, 37 ° C, 5% CO2 incubator culture tank culture.
2. u0026 Nbsp; about 2.5 hours prior to experiment, when the cell density reached 70%, 1 bottle of tritokine 200 u0026 mu; L, the final concentration is 1 u0026 Mu; g / ml, The same amount of PBS (pH 7.4) was added to the 2 type. Co-incubation into the incubator to continue to cultivate for 2.5 hours.
3. u0026 Nbsp; HO33342 and PI Double dyeing identification of three cells

(1) Dyeing: Shake the cells in the bottle with 200 u0026 mu; l to 1.5 mL In the centrifuge tube, HO33342 mother liquor 2 u0026 Mu; L, Pi 20 u0026 Mu; L, stained for 15 minutes.

(2) Take a slide of a slide into a small chamber, from each of the centrifugal tubes 10 u0026 mu; L, add a small indoor cover Hollow slides, fluorescent mirroscopy under high magnification, high allactors, distinguish three cells, and pay attention to the ratio of the triple.




Precautions

1. induced cultured HL-60 cellular time To be accurate;

2. u0026 Nbsp; When the cells were observed under fluorescence microscope, the fluorescence was easily crushed, and it was as fast as possible.
Other
cell death is divided into two different types of apoptosis and necrosis based on its nature, origin and biological meaning. Apoptosis is generally existed in the life community, plays a very important role in biological individuals and survival. It is a combination of cells in a series of sequential sequence under physiological conditions, which is a self-control process that ends the law ends life. Cell apoptosis has identified morphological and biochemical characteristics.
Apoptotic cells are visible to the surrounding cells, and the cell membrane, and the cell membrane is inwarded.Serum, cytosol concentration, endoplasmic network expansion, nuclear lamination of nuclear compression, incubation, endometric network and cell membrane further fused cells into a plurality of complete wrapped apoptosis, apoptosis After being digested by phagocytic cells. During the apoptosis, the contents of the cells do not release the cells without affecting other cells, and thus does not cause inflammatory response.
In biochemical, in the process of most cell apoptosis, endogenous nucleic acid endozyme activation, activity increased. Nuclear DNA randomly is cut off at the connection of nuclear body, degraded into 180-200 bp or its whole number of pieces. If agarose electrophoresis is carried out for nuclear DNA, the characteristics of DNA Ladder (ladder stripes) of 180-200 bp can be displayed.
In contrast, necrosis is cell death that occurred under severe damage conditions. The cells lost the metallic membrane integrity in the early necrosis, and various orpatipotectors expanded, and the contents of the plasma formation released, causing inflammatory response, although the nucleus DNA was also degraded during necrosis, but DNAs in the presence of various lengths Pieces, can't form a trapezoidal stripe, which is disperse. Some mild damage stimulation and some anti-tumor drugs can induce apoptosis, usually these factors can also produce cell necrosis while inducing apoptosis, depending on the severe degree of damage and cell itself Sensitivity to stimulation.