Chromosome specimen preparation of mouse bone marrow cells

Summary

This lab provides an understanding of how mammalian chromosome specimens are made and observations of animal chromosomes. Source: Laboratory Course in Genetics

Operation method

colchicine treatment

Principle

As the hematopoietic stem cells in the bone marrow cells in the limb bones of mice are generating various blood cells and primitive cells with high dividing ability, this material was used in this experiment, and the chromosome specimens were made through the steps of pre-processing, hypotonicity, fixation, preparation, and staining, and many chromosomes in the middle stage of division were observed, which allowed chromosome grouping and phenotyping analysis to be carried out.

Materials and Instruments

Mice
Colchicine Kiemsa Stain Fixative Hypotonic Solution Saline
Balance Centrifuge Constant temperature incubator Microscope

Move

1. Wearing canvas gloves, mice were executed by decapitation method. Immediately, the skin and muscles of the thighs were cut off with scissors, and the femur and tibia on both sides were removed together with the joint head, and the muscles on the joint head were picked out and washed with 2% sodium citrate solution. Cut off the joint head to expose the bone marrow cavity, use a syringe with appropriate amount of 2% sodium citrate solution, insert the needle into the bone marrow cavity, wash the bone marrow with sodium citrate solution into a graduated centrifuge tube, and wash repeatedly until the bone cavity becomes white.

2. Centrifuge the bone marrow cell suspension at 1000r/min for 10min, aspirate the supernatant, add 0.075mol/L KCl 5~8ml, immediately blow with a pipette to make it uniform, and leave it at room temperature for 25min at hypotonic level.

3.1000r/min centrifugation for 10min, remove the supernatant, add 5～8ml methanol-glacial acetic acid (3∶1) fixative along the wall of the tube, immediately blowing and beating evenly, and leave for 30min.

4. Centrifuge the supernatant, leave about 0.1～0.2ml of precipitated cells and supernatant, and mix them with pipette gently and repeatedly to make cell suspension.

5. Take a pre-cooled slide in clean ice water, slightly tilted, use a pipette to suck a drop of the above cell suspension, drop on the slide at the appropriate height above the slide, and immediately blow away and blow the cells on the slide, air dry.

6. After the slides were fully dried, they were stained with phosphate buffer pH 6.8 by mixing 1 part of Giemsa stock solution and 9 parts of phosphate buffer. Material side up, cover the slide with the staining solution, staining for 10~15min, wash away excess staining solution with running water, and then absorb excess water with absorbent paper, drying can be microscopic examination. Stain the slide with carbolic acid magenta for about 10 min.

7. Observe the chromosome preparations after Giemsa staining under low magnification and medium magnification, find out the middle chromosome images with moderate dispersion, and observe them carefully under oil lens to familiarize with the morphology of mouse chromosomes and count the number of chromosomes in bone marrow cells.

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