The time of cloning is generally said to be as early as possible. Because at this time, various hybridoma cells grow vigorously at the same time, competing for nutrition and space, and the cells producing the designated antibodies are likely to be submerged and eliminated. However, the cloning time should not be too early, too early cell traits are unstable, and the number is small and easy to lose.
The cloned positive hybridoma cells, after being cultured for a period of time, also cause some cells to lose the ability to produce antibodies due to cell mutation or loss of specific chromosomes, so that it is necessary to clone or culture again. The number of cloning times is determined by the strength of secretion and the immunity of the antigen. Generally speaking, the number of immunogenic antigen clones can be less, but at least 3 to 5 clones can be stabilized.
There are many methods for cloning, including soft agar cloning, micromanipulation, fluorescence activated separation and limiting dilution.
(a) soft agar cloning
Cloning is achieved by singular growth of individual cells on soft agar, as follows:
1. Add 30 ml of 2.5% agarose, dissolve in a water bath, and transfer to a 45 ° C water bath.
2. 117 ml of complete DMEM solution and 3 ml of 10-fold concentration of DMEM solution were mixed and preheated in a 45 ° C water bath.
3. The agarose was mixed with DMEM solution, which was a complete DMEM solution containing 0.5% agarose, and 75×10 8 spleen cells were added.
4. Add 10 ml to each plate and solidify at room temperature.
5. The cells in DMEM were mixed 1:1 with DMEM-agar, and 2 ml of the cell agar mixture was spread on a solidified plate to cover it all.
6. The mixture was placed in a CO 2 tank at a saturated humidity and cultured at 37 ° C for 10 days.
7. 0.6% agarose was prepared in PBS, dissolved in a boiling water bath, and placed at 45 ° C. A test tube was taken under heat, and 0.1 ml of 25% sheep red blood cells, 0.2 ml of guinea pig complement, and 2.7 ml of 0.6% agarose were quickly added.
8. The clones were covered with a 3 ml agarose-male red blood cell mixture. Incubate in a CO 2 box at 37 ° C for 1 h to 2 h. The hemolysis range of the red blood cells can be screened from the upper part of the clone to screen the anti-sheep red blood cell Ig.
(two) microscope operation
In a 6 cm diameter culture dish, add 1 ml of 1.0×10 8 cell suspension, place 5% CO 2 saturated humidity, place in a 37 ° C incubator for more than 30 min, and place an inverted microscope to find individual cells that are far away from the surrounding area. The mouth (one with a right-angled elbow capillary, one end connected to a one-foot long latex tube, and the mouth is used to control the liquid to enter) is placed horizontally on the liquid surface, and the left and right are slightly moved until the tube is seen, the cells are aligned, the capillary is sucked, and the tube is placed. The cells were transferred to a 96-well plate supplemented with 2.0×10 4 to 5.0×10 4 feeder cells in advance, and after culturing, clones formed by single cells were obtained.
(III) Fluorescence activated separation method
A fluorescence activated cell sorter (Fluorescein Activafed Cell Sorter, FACS) was used. The basic principle is: after the cells are stained by fluorescent antibody, a linear droplet of a single cell is formed through a nozzle, and under the excitation of the light, the fluorescein emits fluorescence, and the signal is received by the photomultiplier tube, and then combined with the cell shape to generate light scattering. The signal is processed by a computer to generate a signal and compared with a predetermined signal. According to the fluorescence intensity and cell size of the cell, the cells are divided into different levels, and deviations occur in the electric field, and are collected in different containers.
(4) Limited dilution method
1. material
(1) Microplates, each well of the plate was cultured on the day before cloning, and the mouse peritoneal cells (ie, feeder cells) were 20,000 to 40,000 per well.
(2) HT medium
2. Method of operation
(1) The antibody-positive well cells were taken out, and a cell suspension was prepared using HT medium. Samples were taken for trypan blue staining and counting.
(2) The cells were diluted with HT medium to a suspension of 200/ml, 40/ml, 20/ml.
(3) The cell suspension was separately seeded into a microplate with a pipette, 0.05 ml per well, and the cell contents were 10 cells/well, 2 cells/well, 1 cell/well and 0.5 cells/well, respectively.
(4) 5% CO 2 saturated humidity, cultured at 37 ° C.
(5) Observe the growth of the clones with an inverted microscope every day, select a well with only one colony, and discard more than two wells with no cell growth.
(6) After the clone is multiplied, the culture solution antibody is measured when 1/3 to 1/2 of the bottom of the well is covered.
(7) The antibody-positive well cells are transferred to a tissue culture flask with a feeder layer, and can be detached from the feeder cells for 2 to 4 generations to construct a cloned strain.
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