First, the principle

Cell fusion, a process in which two or more cells are combined to form a single cell under natural conditions or by artificial means (biological, physical, chemical). Artificially induced cell fusion was developed as an emerging technology in the 1960s. Cell fusion technology is currently widely used in various fields of cell biology and medical research because it not only produces the same kind of cell fusion, but also produces interspecific cell fusion.

There are many types of inducers of cell fusion. The most commonly used are inactivated Sendai virus, polyethyleneglycol (PEG) and electrical pulse. Polyethylene glycol is the most widely used because it is easy to obtain, simple, and stable. The mechanism of PEG fusion is not fully understood, and it may cause structural rearrangement of acyl bonds and polar groups of phospholipids in cell membranes.

Second, the method

(1) Take fresh chicken blood and make a 10% suspension with 0.85% physiological saline.

(2) Weigh 0.5 g of PEG (MW = 4,000) into a test tube, melt it on an alcohol lamp, and quickly add 0.5 ml of preheated Hanks solution to make a 50% PEG solution. Place in a 37 ° C water bath for use.

(3) Take 1 ml of the above 10% chicken red blood cell suspension into a centrifuge tube, add 5 ml of Hanks solution and mix, then centrifuge at 1,000 rpm for 5 minutes, carefully discard the supernatant, and use the finger bomb method to bounce the cell mass. .

(4) Take 0.5 ml of the above 50% PEG solution, add dropwise to the chicken red blood cell suspension within 1 minute, and gently shake while mixing. After all the PEG was added, it was allowed to stand for about 1 minute. This entire process is required to be carried out in a 37 ° C water bath.

(5) 9 ml of Hanks solution was slowly added dropwise to terminate the action of PEG, and allowed to stand in a 37 ° C water bath for 5 minutes.

(6) After centrifuging and discarding the supernatant, take a drop of the fused cell suspension and attach a microscopic examination.

Third, the results of observation

At high magnification, it can be seen that two or more chicken erythrocyte membranes fuse together to form a heteronuclear somatic cell. Care should be taken to identify fused cells and overlapping chicken red blood cells.

Fourth, the calculation of the fusion rate

Randomly count 200 cells (including fused and unfused cells) at high magnification, dividing the number of nuclei of the fused cells (cells containing two or more nuclei) by the total number of nuclei (including fusion and unfusion) The nucleus) gives the fusion rate.

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