Preparation of myeloma cells
The manner in which pre-fusion myeloma cells are maintained is most important for successful hybridomas. The goal is to keep the cells in logarithmic growth for as long as possible, and certainly not less than 1 week before fusion. Cryopreserved cells will be in a state suitable for fusion after 2 weeks of resuscitation, and long-lasting myeloma cells may recover at least for a few days. Myeloma cells that were logarithmicly grown in the laboratory were maintained in medium containing 10% calf serum by inoculating 10-fold serially diluted myeloma cells in 6 flasks of 5 ml medium. After 1 week, a bottle of cells that were quite dense and not long was re-transplanted. A typical doubling time is 14-16 hours.
The preparation method of myeloma cell suspension is as follows
1 Bone marrow cells were expanded and cultured 48-36 hours before the fusion (generally, 2-3 bottles of 100 ml culture flasks were prepared in a fusion assay of 96-well plates).
2 On the day of fusion, the cells were gently blown from the vial with a curved dropper and collected in a 50 ml centrifuge tube or fusion tube.
Centrifuge at 31000 r/min for 5-10 minutes and discard the supernatant.
4 Add 30 ml of incomplete medium and wash once with the same method. The cells were then resuspended in 10 ml of incomplete medium and mixed.
5 Take the myeloma cell suspension, add 0.4% trypan blue dye solution for live cell counting and use. When the cells were counted, 0.1 ml of the cell suspension was added to 0.9 ml of trypan blue dye solution, mixed, and counted using a hemocytometer. The formula for calculating the number of cells is: number of cells per ml = 4 large square cells × 105 / 4; or number of cells per ml = 5 square cells × 106 / 2
Preparation of spleen lymphocytes
The BALB/c mice that had been immunized were taken, the eyeballs were removed, and the serum was separated as a positive control serum at the time of antibody detection. At the same time, the mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes, fixed on the dissection platen, and then the left abdomen skin was opened. The spleen was seen, the eye scissors were cut, and the sterile surgical scissors were used in the ultra-clean table. The peritoneum was cut open, and the spleen was taken out and placed in a dish containing 10 ml of incomplete medium, gently washed, and the surrounding connective tissue was carefully peeled off. The spleen was transferred to another dish containing 10 ml of incomplete medium, and the spleen was gently squeezed with a curved tweezers or a curved needle on a 1 ml syringe (the spleen could also be squeezed with a syringe core) to allow the spleen cells to enter the plate. Incomplete medium in the medium. Sip several times with a pipette to make a single cell suspension. To remove large agglomerates from the spleen cell suspension, a 200 mesh copper mesh can be used for filtration. The spleen cell suspension was harvested, centrifuged at 1000 r/min for 5-10 minutes, washed with incomplete medium for 1-2 times, then resuspended in 10 ml of incomplete medium, and the suspension was added to add phenol blue. The dye solution is counted as a live cell count. Usually, 1 x 108-2.5 x 108 splenocytes can be obtained per mouse, and 5 x 108-10 x 108 splenocytes can be obtained from the spleen of each rat.
Preparation of feeder cells
In the selective culture process after cell fusion, since a large number of myeloma cells and spleen cells die successively, at this time, single or a small number of scattered hybridoma cells are not easy to survive, and it is usually necessary to add other living cells to breed, and this is added. Living cells are called feeder cells. The mechanism by which feeder cells promote other t cell hybridoma proliferation is unclear. It is generally believed that they may release non-species-specific growth stimulating factors to provide the necessary growth conditions for hybridoma cells; or to satisfy the cell density of neonatal hybridoma cells. Dependence.
Commonly used feeder cells are thymocytes, normal spleen cells, and peritoneal macrophages. Among them, the source and preparation of mouse peritoneal macrophages are convenient, and the function of phagocytosis to eliminate dead cells and fragments thereof is the most common. The preparation method is as follows:
After the mice were killed, the surface was disinfected and fixed according to the above method of collecting mouse spleen cells, the abdominal skin was lifted from the posterior abdomen with a sterile scissors, and the peritoneum was exposed. Wipe the peritoneum with an alcohol cotton ball. Inject 10 ml of incomplete medium into the abdominal cavity with a syringe, taking care to avoid penetrating the intestine. Fix the syringe in the right hand, leave the needle in the abdominal cavity, gently massage the abdomen with an alcohol cotton ball for 1 minute, then aspirate the injected culture solution. Centrifuge at 1000r/min for 5-10 minutes and discard the supernatant. The precipitated cells were first suspended in 5 ml of HAT medium, and HAT medium was added according to the cell count to make the cell concentration 2 × 105 / ml, and set aside. Generally, for macrophages, 96-well plates require 2 x 104 cells per well, and 24-well plates require 105 cells per well. Each mouse can obtain 3-5 x 106 cells, so one mouse can be used for two 96-well plate feeder cells. The feeder cells can also be prepared and cultured 1-2 days before cell fusion, such that the bottom of the culture plate is first covered with a layer of feeder cells. The above suspension was added to a 96-well plate at 0.1 ml per well (equivalent to 2 drops), and then cultured in an incubator at 37 ° C 6% CO 2 .
Cell fusion and selective culture of hybridoma cells
There are many reports on cell fusion procedures, and one is commonly used in the author's laboratory.
1 Mix 1×108 splenocytes with 2×107-5×107 myeloma cells SP2/0-Ag14 in a 50 ml fusion tube, add incomplete medium to 30 ml, and mix well.
Centrifuge at 21000 r/min for 5-10 minutes and absorb the supernatant as much as possible.
3 Tap the bottom of the fusion tube on the palm to make the precipitated cells loose and even; preheat in a 40 °C water bath.
4 Using a 1 ml pipette for about 1 minute (optimal time is 45 seconds), add 1 ml of 50% PEG (pH 8.0) preheated to 40 ° C, and gently stir while adding.
5 Using 20 ml of a pipette, add 20-30 ml of incomplete medium preheated to 37 ° C in 90 seconds; let stand at 20-37 ° C for 10 minutes.
61000r/min 5 minutes; discard the supernatant.
7 Add 5 ml of HAT medium, gently pipette the precipitated cells, suspend and mix, and then add HAT medium containing peritoneal macrophages to 80-100 ml.
8 well 96-well cell culture plates were used, 0.10-0.15 ml per well; 24 well plates were dispensed, 1.0-1.5 ml per well; then the plates were incubated at 37 ° C in a 6% CO 2 incubator.
After 95 days, 1/2 medium was exchanged with HAT medium.
The HAT medium was exchanged with HT medium after 107-10 days; (normal complete medium can be used after the 14th day). The growth of hybridoma cells is often observed, and the supernatant is aspirated for antibody detection until it reaches a cell bottom area of 1/10 or more.
Hybridoma cell screening
Hybridoma cells can be screened about 2 weeks after fusion, that is, the hybridoma pores secreting the desired antibody are selected from a plurality of wells, which are also commonly referred to as antibody detection. There are many methods for antibody detection, usually based on the antigen being studied and laboratory conditions. However, the antibody detection method for hybridoma screening must be fast, prepared, simple, and convenient for processing a large number of samples at a time. Because there are often hundreds of samples that need to report results in just a few hours in order to determine the trade-offs of human hybridoma cells. Therefore, the principle of using antibody detection methods is fast, sensitive, specific, reliable, low cost and labor saving. In general, antibody detection methods must be established prior to fusion and overcome possible problems. Another important issue is the "kinetic range" required for antibody detection methods, ie the ratio of the strongest to the weakest signal above the background, depending on whether the antigen used is pure. For example, a hybridoma human anti human antibody is directed against a purified protein antigen, and 100% of the antigen participates in the reaction, and a positive/negative discrimination system is sufficient. On the other hand, if the hybridoma antibody is directed against a small amount of protein antigen on the cell surface, the detection system may need to be able to detect weak signals, and the kinetic range should be at least 10:1, preferably 100:1. In addition, the choice of detection method is also affected by the type of hybridoma antibody desired and the intended use. Antibodies that bind to complement can be selected using a cytotoxic reaction based assay. For hybridoma antibodies that bind to protein A, a binding protein A assay is used.
Cloning of hybridoma cells
The positive hybridoma cells obtained from the original wells may be derived from two or more hybridoma cells, and thus the antibodies they secrete are heterogeneous. In order to obtain a completely homogeneous monoclonal antibody, hybridoma cells must be cloned. On the other hand, the early stage of hybridoma cell culture is unstable, and some cells lose part of the chromosome and may lose the ability to produce antibodies. In order to remove this part of the cell which no longer secretes the antibody, a monoclonal hybridoma cell line (also referred to as a subcloning) which is secreted by the secreted antibody is obtained, and cloning is also required. In addition, long-term liquid nitrogen cryopreserved hybridoma cells may still lose their ability to secrete antibodies after resuscitation, and therefore should be cloned to detect antibody secretion. Usually, after obtaining a hybridoma against a predetermined antigen, 2-3 clonings are required continuously, sometimes multiple times. By cloning is meant the entire culturing process in which a single cell is vegetatively propagated to obtain the cell mass. There are many methods for cloning, such as limiting dilution, soft agar method, single cell micromanipulation, monoclonal cell group micromanipulation, and fluorescence activated cell sorter (FACS) separation.
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