Cell Technology Topics: Animal Cell Fusion

Cell fusion refers to the process of combining two or more cells into one cell. Under natural conditions, spontaneous fusion occurs in both in vivo and in vitro cultured cells. The artificial method of inducing cell fusion began in 50 years, and now this technology has become an important means of studying cell genetics, cellular immunity, tumor and cell engineering. Under the action of inducers (such as Sendai virus, polyethylene glycol), the cells fused to each other agglutinate, and then a series of changes in plasma membrane components occur at the plasma membrane contact, mainly the breaking and rearrangement of certain chemical bonds. The two plasma membranes are opened to form a binuclear or multinucleated cell (referred to as a homonuclear or heteronuclear body at this time). Through mitosis, the nucleus fuses to form hybrid cells.

Laboratory supplies

1. Apparatus microscope centrifuge balance tube syringe fine dropper slide, cover slip

2. Reagent 50% polyethylene glycol (PEG): Weigh a certain amount of PEG (MW=4000) into a graduated tube and heat it in an alcohol lamp or boiling water. When cooled to 50 ° C, add an equal volume of GKN liquid preheated to 50 ° C to mix.

Alsver solution: glucose 2.05 g sodium citrate 0.8 g NaCl 0.42 g, and the distilled water was added to 100 ml.

GKN solution: NaCl 8 g, KCl 0.4 g, Na ₂ HPO ₄ · 2H ₂ O 1.77 g, NaH ₂ PO·2H ₂ O 0.69 g, glucose 2 g, phenol red 0.01 g, dissolved in 1000 ml of re-distilled water.
0.85% NaCl solution

3. Materials one year old cock venous blood

Experimental content and method

1. Take 2ml of Alsver solution with a syringe, then take 2ml of chicken blood from the subpteral vein, inject into the test tube, add 6ml of Alsver solution, mix and store in 4°C refrigerator for 3-4 days.

2. Take 1 ml of "1" solution in a centrifuge tube, add 4 ml of 0.85% NaCl solution, mix well and centrifuge at 1200 r/min for 5 minutes.

3. Remove the supernatant. Repeat "2" twice more and centrifuge for 10 minutes for the last time.

4. Add 1 ml of GKN solution to the sedimented blood cells and mix to make a cell suspension. (GKN can be added to adjust the dilution to make 30,000 to 40,000 red cells per cubic millimeter).

5. Add 6-8 drops (about 0.5ml) of 50% PEG solution to the “4” solution, mix quickly, and take a microscopic examination at room temperature for 2~3 minutes. Pay attention to different degrees of fusion during observation. Usually divided into five stages. 1 two cell membrane contact, adhesion; 2 cell membrane formation perforation; 3 cytoplasmic communication of two cells; 4 channels expanded, two cells joined together; 5 cells completely merged to form a circular cell containing two or more nuclei.

6. Calculate the fusion rate.