Due to the high activity of maturation promoting factor (MPF), the fusion of M-phase cells with interphase cells can cause the interphase cells to show morphologic changes similar to those of mitosis: chromatin condensation, disintegration of the nuclear membrane, and disappearance of the nucleolus, etc. This kind of induced chromosome formation in interphase cells is called prematurely condensed chromosome (PCC). This kind of chromosome formed in interphase cells after induction is called prematurely condensed chromosome or precocious condensed chromosome (PCC).
Principle
The principle of chromosome pre-condensation specimen preparation is to fuse M-phase cells with G1-phase cells, then the chromatin of G1-phase cells condenses into a single thin and long pre-condensed chromosome visible under the light microscope, i.e., G1-PCC.The length and thickness of the chromosome are closely related to the position of the cell in G1 phase: the chromosome in the early G1 phase is short and thick, while the chromosome in the late G1 phase is thin and long. S-phase cells are at the stage of DNA replication, and a large number of replication units start replication at different times, so the replicating chromatin is highly de-helicalized, which is not visible under the light microscope, but only the part that has not yet replicated or replicated and then re-agglomerated.M-phase cells induce S-phase cells to have chromatin agglomerations in the form of powders or crushed granules, which can be seen under the electron microscope: the granules are the parts of the chromatin that are highly helicated, and are connected by slender chromatin filaments between granules. The morphology of G2-PCC is close to that of M-phase chromosomes, except that the degree of helicalization is lower, so its staining is shallow, elongated, and the sister chromatids are close to each other.
Appliance
Chromosome advance agglutination specimens are commonly used in clinical practice for cell cycle analysis, study of the microstructure of chromosomes in normal and tumor cells, study of chromosome damage and repair effects caused by a variety of factors, and diagnosis and prognosis of hematological diseases.
Operation method
Preparation of chromosome advance agglutination specimens
Principle
The principle of chromosome pre-condensation specimen preparation is to fuse M-phase cells with G1-phase cells, then the chromatin of G1-phase cells condenses into a single thin and long pre-condensed chromosome visible under the light microscope, i.e., G1-PCC.The length and thickness of the chromosome are closely related to the position of the cell in G1 phase: the chromosome in the early G1 phase is short and thick, while the chromosome in the late G1 phase is thin and long. S-phase cells are at the stage of DNA replication, and a large number of replication units start replication at different times, so the replicating chromatin is highly de-helicalized, which is not visible under the light microscope, but only the part that has not yet replicated or replicated and then re-agglomerated.M-phase cells induce S-phase cells to have chromatin agglomerations in the form of powders or crushed granules, which can be seen under the electron microscope: the granules are the parts of the chromatin that are highly helicated, and are connected by slender chromatin filaments between granules. The morphology of G2-PCC is close to that of M-phase chromosomes, except that the degree of helicalization is lower, so its staining is shallow, elongated, and the sister chromatids are close to each other.
Materials and Instruments
Equipment. Move The basic process of preparing chromosome advance agglutination specimens can be divided into the following steps: (4) Add 5 ml of Hanks solution and shake the culture flask repeatedly for 3~5 min, or gently blow the cell layer with a pipette. Since the M phase cells are spherical, the contact area with the wall of the bottle becomes smaller, and they are easy to be detached and suspended in the culture medium. Transfer the cell suspension into a 5 ml graduated centrifuge tube and count the cells. Caveat (1) In order to ensure the fusion rate, the supernatant should be discarded as much as possible before adding PEG; when adding 50% PEG dropwise, it should be added slowly, drop by drop, and be careful to mix gently during the process.(2) After adding PEG and standing for 1 min, dilute 10 times with culture medium, and do it gently. Incubate at 37 ℃ for a longer period of time (30~60 min), and generally a high proportion of PCC can be obtained. For more product details, please visit Aladdin Scientific website.
① Ordinary optical microscope
② Ordinary low-speed centrifuge
③ CO
2
Temperature-controlled incubator
④ Ultra-clean bench
⑤ 5 ml graduated centrifuge tube, disposable burette, slide, coverslip, cell counting plate reagents.
Materials: ① HeLa cells
① HeLa cells
① HeLa cells ② PEG (M, = 4000) (50%)
① HeLa cells ② PEG (M, = 4000) (50%) ③ RPMI-1640 culture medium
③ RPMI-1640 culture medium ④ Calf serum
⑤ Hanks liquid
⑥ Colchicine (10 μg/ml) ⑦ Trypsin (0.5 μg/ml)
⑦ Trypsin (0.25%)
⑧ KC1 (0.075 mol/L)
⑨ Methanol-glacial acetic acid (3:1) (freshly prepared)
⑩ Jimsa's stain
1 . Preparation of M-phase cells
(1) Passage of the cells was performed in the usual way.
(2) When the cells were cultured for 2~3 d, and the cells were growing vigorously and in the logarithmic growth phase, colchicine (final concentration of 0.2~0.5 μg/ml) was added, and continued to be cultured for 3~4 h in a CO2 constant temperature incubator.
(3) Gently pour off the culture medium, and wash the cells with 5 ml of Hanks' solution for 2 times, and discard the dead cells, cellular debris and Hanks' solution.
Add trypsin solution with a final concentration of 0.25% to the wall cells collected from M-phase cells, digest for 2~3 min, discard the digested solution, add 5 ml of Hanks solution, blow the cell layer repeatedly with a pipette and transfer the cell suspension into 5 ml graduated centrifuge tubes for counting.
3 . Cell fusion
(1) Mix M-phase cells and interphase cells at a ratio of 1:1 (about 106 cells each). (1)Mix M-phase cells and interphase cells in a 1:1 ratio (about 106 cells each) in a 5 ml centrifuge tube, centrifuge at 800 g for 5 min, and discard the supernatant. The supernatant was washed and centrifuged twice with 5 ml of Hanks solution, and the supernatant was discarded as much as possible.
(2) Gently flick the bottom of the tube to loosen the precipitate, pre-warm it in a water bath at 37 ℃ (39 ℃ is better), slowly add 0.5~1 ml of pre-warmed 50% PEG solution at 37 ℃ drop by drop, gently mix with a pipette as it is added (you can also gently shake the mixture during the process of addition and then gently blow with a pipette at the end of the mixing process). 37 ℃ leave it for 1 min.
(3) Add 5 ml of serum-free RPMI-1640 culture medium (the first 1 ml should be serum-free). (3) Add 5 ml of serum-free RPMI-1640 culture medium (the first 1 ml should be added slowly drop by drop), mix well (dilute to terminate the effect of PEG solution). 800 g centrifugation for 5 min (centrifugation can be left at 37 ℃ for 5~10 min), discard the supernatant.
(4) Add RPMI-1640 growth medium containing calf serum, gently blow and mix well to suspend the cells. 37 ℃, 5% CO2 incubation for 30~60 min.
4 . Preparation
Remove the above cells, centrifuge at 800 g for 8 min, and discard the supernatant. Prepare the chromosome specimen according to the conventional method. Observe the results after staining with Giemsa's stain.