Chromosome specimen preparation experiments for in vitro cultured cells

Summary

The source of this experiment is the official website of the Fourth Military Medical University

Operation method

Chromosome specimen preparation experiments for in vitro cultured cells

Materials and Instruments

cytoplasm

Move

1. When cells cultured in vitro were observed to have a high number of dividing cells (round, shiny cells 24 h after passaging) under an inverted microscope, colchicine solution was added at a final concentration of 0.3 μg/ml culture solution;2. Continue incubation for 3h;3. Trypsin digestion was performed to collect the cells into centrifuge tubes;4. Centrifuge at 1000 rpm for 10 min and remove the supernatant;5. Wash with Hanks solution, centrifuge, and remove supernatant;6. Hypotonic treatment: Add 8ml of 0.075mol/L KCl solution, place in a 37℃ constant temperature water bath for 30min, and blow slightly with a pipette (as above);7. Fixation and preparation of films such as peripheral blood chromosomes.

Caveat

1. Colchicine treatment time is too long, the division of cells, short chromosomes; on the contrary, less and slender, so the concentration of colchicine and time should be accurately mastered.

2. The fixative should be prepared temporarily before use.

3. The slides must be clean, otherwise the chromosomes will not be well dispersed.

4. Low chromosome division index: the patient is in a very period (radiotherapy, chemotherapy); poor nutritional composition of the medium; medium pH is low or high; PHA is too much or not enough; the quality of calf serum is not high; the temperature of the incubator is low; too short colchicine treatment time.

5. The fresher the inoculated blood sample, the better.

6. If blood samples are found to be agglutinated during the incubation process, the culture bottle can be gently shaken to disperse the clots and continue to be put back into the 37℃ incubator for incubation.

Common Problems

Carnoy's fixative:

The important properties of a fixative are the ability to rapidly penetrate cells, fix them and maintain the structural integrity of the chromosomes, as well as the ability to enhance the basophilicity of the chromosomes to achieve excellent staining results. It is generally difficult for a single fixative to meet these requirements, so a mixture of two fixatives is used in the experiments. Because Carnoy first used methanol and glacial acetic acid mixture and called it Carnoy's fixative. Carnoy's fixative (methanol: glacial acetic acid = 3:1) each time to use before the temporary preparation, a long period of time to affect the effect of the fixation, fixation time of 15min to 24h, refrigerator, room temperature can be. If necessary, the ratio of methanol and glacial acetic acid can be changed. An increase in the ratio of glacial acetic acid is good for cell expansion and chromosome spreading, but it is easy to lead to cell rupture and chromosome dispersion.

Hypotonic solution (hypotonic solution)

Hypotonic solution refers to the osmotic pressure and ionic strength are lower than the normal cell physiological conditions of the solution, the osmotic pressure is lower than the tissue fluid, and mixed with peripheral blood, the water quickly into the cell, so that it swells, or even rupture, to obtain well-dispersed chromosome division image. Common hypotonic solutions: water, hypotonic sodium citrate or sodium chloride, potassium glycerophosphate (0.65 mol/L), potassium chloride (0.075 mol/L), and others. The effectiveness of hypotonicity depends on the chemical composition of the hypotonic fluid, the temperature of the hypotonicity and the treatment time. Hypotonic treatment is by virtue of reverse osmosis to make the cell swelling chromosomes spread, at the same time can make the karyorrhexis material adhering to the chromosomes scattered, so that all chromosome morphology can be observed in a plane. Laboratory generally choose 0.075mol / L KCl for hypotonic solution, the advantages are: ① chromosome outline is clear, can be stained, staining time is short. ② When used for banding staining, it can fully display the banding characteristics. The hypotonic treatment is 37℃, 25-30min, subject to the pre-experimental conditions.

Giemsa staining solution

Preparation of Giemsa stock solution: weigh 0.5g of Giemsa powder, add a few drops of glycerol and grind, then add glycerol (so that the total amount of glycerol added is 33 ml). keep warm at 56℃ for 90-120min. add 33ml of methanol and store in a brown bottle. When used, dilute with PBS as required, usually 10 times diluted.

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