JRD248 apoptosis experiment

Cell apoptosis experiment: The cells within the human body are destined to die, some death is physiological, some death is pathological, to In this respect, people have known that there are two ways to death, namely, cell necrosis and apoptosis (apoptosis). Apoptosis refers to a basic biological phenomenon of cells, which is a basic biological phenomenon of cells, which is a basic biological phenomenon of cells, which is a basic biology of cells. Role. Apoptosis is the process of multi-gene strict control. These genes are very conservative between seeds, such as BCL-2 family, Caspase family, carcinoma, such as C-Myc, tumor cancer gene P53, and the like. The disorder of apoptosis may have a direct or indirect relationship with many diseases. Such as tumors, autoimmune diseases, etc., many factors that can induce apoptosis, such as rays, drugs, etc.
Apoptosis and necrosis difference:
Necrosis is the death process of cells that cause cell disorderly variation in cells. It is characterized by cell bloating, cell membrane rupture, cell contents, slower changes, DNA degradation is not sufficient, causing local severe inflammatory response.
Apoptosis is a death process of a cytoplasmic physiological pathological stimulus signal, a change in environmental conditions or a dequinating process of response or ordering of mitigatory injury. The changes in cells and tissues are significantly different from necrosis.


Morphological changes: Morphological observation of changes in apoptosis experiments are multi-stage, often involving individual cells, even a small portion of cells are also asynchronous. First, the cell volume is reduced, the connection disappears, detached from the surrounding cells, then the cytoplasmic density increases, the mitochondrial film potential disappears, the permeability change, the cytosol C to the cytoplasm, the nucleus concentration, nuclear membrane nuclear crush DNA degradation becomes about 180 bp-200 bp fragment; the cell membrane has a small cubilated formation, and the inner phosphatidylisine is flipped to the surface of the membrane. The membrane structure is still complete, and the residual segmentation of apoptotic cells will be wrapped in several apoptosis. The body, no contents of the surrounding, so there is no surrounding inflammatory response, apoptotic body can be quickly swallowed by the surrounding or non-special phagocytic cells.
A, morphology
Principle: Generally, the progression of apoptosis is judged by the morphological changes of cell nuclear staining. Common DNA-specific dyes are: HO 33342 (Hoechst 33342), HO 33258 (Hoechst 33258), DAPI. The combination of the three dyes and DNA is non-embedded, mainly in the A-T base region of DNA. Ultraviolet light is excited to launch brightBlue fluorescence. The morphological changes of cell nuclear staining in the process of apoptosis are divided into three phases: the cell nuclear nucleus of the phase I is corrugated or creased, and partial chromatin has a concentrated state; the chromatinic height of the IIA nucleus Condensed, marginalized; cell nuclear cracking in the IIB is broken, producing apoptotic body.
Advantages: Simple, convenient, low
Disadvantages: very subjective, very dependent on the experiment of the experimenter
B, DNA content

principle: Cell apoptosis, nucleic acid Endozyme activation, leading to DNA breakage, which is the characteristic expression of apoptosis, also for FCM identifying apoptotic cells. During the cell membranes, vulnerabilities occurred on cell membranes in cell membranes, sediment DNA released from intracellular membranes, which made DNA diploids below normal cells. Approximately the propidium ( pi) dyeing analysis, it can appear u0026 ldquo; sub-diploid u0026 rdquo; peak, ie, apoptosis peak (APO peak) according to APO peaks The percentage of apoptotic cells can be measured.
Advantages: This method is simple and easy, which can determine apoptotic specimens in large batch, and can also simultaneously analyze the cell cycle position of cells.
Disadvantages: DNA Content assay in detecting apoptosis is that its specificity and sensitivity are not high. The specificity is not high because of APO peak represents a group of cell groups, including apoptotic cells, mechanical damage cells, low DNA cells or different chromosome structures, DNA combined with fluorescent dye The amount is small. Further, when the non-fixed cells are dissolved in the low-permeable solution, a large number of nuclear debris can occur, at which time APO the number of cells of the peak represents the number of nuclear debris, does not represent the number of apoptotic cells. The reason for the sensitivity is that there is only DNA u0026 NBSP in the early stage of apoptosis; the fracture point appears, but DNA u0026 NBSP is not appeared; the fragment is largely lost, so the method cannot detect early apoptotic cells and the S M u0026 NBSP. The apoptotic cells, because the actual content is not less than DNA contained in diploid cells, so the method should be combined with other morphological or biochemical methods, in order to more accurately analyze the apoptotic state of the cells. .
C, Annexinv / Pi

Principle: PS Transfer from the inner side of the cell membrane to the outside induced by apoptosis induction, may be identified as an identification mark of the immune system. Annexinv, a calcium-dependent phospholipin binding protein, can be specificly binding to PS outside the membrane, and then detect by simple color production or light-emitting systemMeasurement. Since this is an endocyte detection of apoptosis (suspended cells and adaptive cells), the development stage of apoptosis can be marked in combination with DNA dyes or other advanced detection methods.
Advantages: Cell apoptosis, PS exposed earlier than DNA fracture occurred, so the method was more sensitive, and the method did not need to fix cells, avoiding PI Fixed cells Excessive fragmentation and TUNEL France DNA u0026 NBSP due to fixed DNA the fragment is lost, so more time, the results are more reliable, and is currently an ideal apoptosis quantitative detection method.
Disadvantages: It is better to detect early apoptosis, good use of other methods of detection of advanced apoptosis, and is high for testing reagents.
D, mitochondrial film potential
Principle: The mitochondrial transfer film potential induced by apoptosis is changed, resulting in a change in membrane penetration. Mitosensortm, a cationic stain, which is very sensitive to this, showing different fluorescent stains. In normal cells, it forms aggregates in the mitochondria, which sounds a strong red fluorescence. In the apoptotic cell, it is present in the cell fluid in the form of a monomomite in the form of a single body, and green fluorescence is emitted in the cell. These two different fluorescent signals can be clearly resolved with fluorescent microscopy or flow cytometry.

Advantages: The detection method of fluorescence is used to enlarge apoptotic signal and high sensitivity.
Disadvantages: Changes of mitochondrial membrane potentials caused by apoptosis or other causes.
E, TUNEL

Principle: Due to endogenous nucleic acid endozyme activation, cells themselves chromatin or DNA cut, and produced with DNA breakpoint The same 3 u0026 rsquo; Hydroxy end, TDT biotinylated DUTP label to 3 u0026 RSQUO; hydroxy-based end, through ooexein 2Fitc system, the DNA breakpoint parts can be specifically fluorescence Cells. However, due to the breakdown of DNA the labeling process involves a variety of factors, the negative results of the end mark do not represent the completeness of the DNA chain, the problem should be ruled out, such as TDT enzyme activity loss, etc. Many influencing factors. Therefore, TDT end marker method identifies the simultaneous positive and negative control group to obtain reliable results.
Advantages: TDT end marker method is a method of identifying apoptotic cells
Disadvantages: The labeling process is more complicated, involving a variety of factors, the experimental failure is subject to the quality of the reagent, and the operational technique has a greater impact.
F, Caspase-3/7
Principle: caspase-3 is the main end shear enzyme during apoptosis. The caspase-3 is in the form of enzymatic (32 kD) in the form of cytoplasm, and in the early stage of apoptosis, it is activated, activated CASPase-3 Two large subunits (17kd) and two small subunit (12kd) are composed. Caspase-3 The main substrate is a polymer (ADP-Ribose) polymerase PARP (Poly (ADP-Ribose) polymerase, the enzyme is repaired with DNA, and gene integrity monitoring is related. At the start of apoptosis, 116 kD PARP cuts 31 kD and 85 kD in two fragments between ASP216-GLY217, separating two zinc fingers in PARP and DNA binding to the carboxy end of the carboxy end, cannot Play a normal function. As a result, the activity of Ca2 + / Mg2 + dependent nucleic acid endonuclease affected by PARP is increased, and DNA between lysis of the nucleus, causing apoptosis. Caspase 3/7 activity and expression can be detected by Western Blot, Enzymatic Activity Test, Flow Cytokinetics.
Advantages: Caspase 3/7 activity detection, detectable methodology is wide, high sensitivity.
Disadvantages: Need to detect and verify the downstream PARP.
G, PARP

Principle: PARP, POLY (ADP-RIBOSE) polymerase, is located in the nucleus, and the DNA repair is closely related to stress conditions. An enzyme. PARP can be cut by multiple caspase in vitro, in the body is the main shear object of Caspase 3. For human PARP, after Caspase is cut between ASP124 and GLY 215, the PARP carboxyl end of the catalytic domain (89 kD) and the DNA binding domain (24kd) of the amino terminal are separated, thereby causing PARP to lose its enzyme activity. PARP is very important for cell stability and survival, and PARP loss enzyme activity accelerates the instability of cells. PARP shear is considered an important indicator of apoptosis, and is also generally considered to be CASPase 3 activation indicators.
Advantages: Detection method is routinely credible, apoptosis specifically
Disadvantages: no apoptosis

service description:
1. apoptosis detection Can be recommended by our customers, after the customer choice;
2. apoptosis experiments involved in cells, drugs, can be provided by customers, or view the company's product library, can also be purchased by us;
3. Apoptosis Expedition2. Select 2 different methods to verify each other.
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Experimental report

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