Sister chromatid differentiation staining experiment

Sister Chromatid Differential Staining (SCD) is a technique developed in the 1970s that allows the two chromatids of the midtelomeric chromosome to be colored differently, allowing the observation of Sister Chromatid Exchanges (SCEs), which is the phenomenon where two chromatids exchange nucleotide sequences during DNA synthesis. Source: Tissue Culture and Molecular Cytology Techniques. (Beijing Publishing House)

Operation method

Sister chromatid differentiation staining experiment

Principle

Cells are cultured in medium containing 5-Bromodeoxyuridine (5-Bromodeoxyuridine: BUdR), which can act as a precursor for nucleotides to replace the thymidine nucleoside (Thymidine: TdR) doped into newly synthesized DNA as the cells undergo DNA synthesis. During the first cell cycle, each chromosomal DNA double strand of the midtelomeric chromosome is doped with BUdR (TB-TB) due to the mechanism of semiconserved replication. However, in the second cycle, a difference in chemical composition occurs: only one of the two chromatids of the mid-stage chromosome retains half of the old BUdR-free strand, while the other contains BUdR (TB-BB) in both double strands, and when stained with a dibenzimidazole derivative fluorescent dye, Hoechst 33258, the TB strand emits a stronger fluorescence, and the BB strand has a fluorescence-quenching The BB chain has a quenching effect on the fluorescence and emits weak light. Due to the rapid disappearance of Hoechst 33258 fluorescent staining, a permanent specimen method was established to observe SCD. The commonly used methods are thermal phosphate treatment plus Giemsa staining and fluorescent dye plus Giemsa staining (FPG).The TB chain has a strong affinity for Giemsa dye and a poor affinity for the BB chain, which gives results consistent with staining with fluorescent dyes, but results in different shades of differential staining.

Materials and Instruments

Cells Specimens
BUdR NaOH colchicine NaH2PO4 Giemsa Sorensen SSC

Move

I. Preparation of BUdR storage solution

BUdR 5 mg (first dissolved with 0.5 ml of 1 N NaOH), the Then add distilled water to 5 ml. Then add distilled water to 5 ml, that is, 1000 μg / ml of storage solution, because BUdR will be decomposed when exposed to light, the storage solution should be protected from light and frozen storage.
BUdR Admixture and Preparation Procedure
1. Preparation of BUdR culture solution

Prepare a culture solution containing BUdR (final concentration of 3-10 μg/m). 2;
2. BUdR Admixture

If using passaged cells, after the last passaging, change the culture medium with BUdR-containing culture medium; If human peripheral blood cells are used, culture with BUdR-containing culture medium at the beginning (culture flasks are placed in special black (the culture flasks are placed in special black wooden boxes, black cloth or wrapped in black paper) and incubated at 37 ℃. 3;
3. Discontinue mixing and preparation

Allow the cells to go through two cell cycles (48 or 72 hours for human peripheral blood cells). 4;
4. add colchicine → preparation (same as other chromosome preparation methods).
Sister chromatid differentiation staining
1. Method I

(1) Aging

Aging the mid-stage chromosome specimen for 1~2 days;
(2) Pretreatment

Place the specimens in 1 M _NaH2PO4 solution (pH adjusted to 8.0 with NaOH) preheated to 85-89 ℃ for 15 minutes. (pH adjusted to 8.0 with NaOH) for 15 minutes;
(3) Preparation

Then rinse gently with warm evaporated water, rinse with distilled water, dry, stain with Giemsa's solution for 10 minutes, dry, and stain with dimethyl acetate. Giemsa staining for 10 minutes, drying, xylene transparency, and sealing.
2. Method 2 (FPG method)
(1) Specimen

Specimen of intermediate chromosome with BUdR;
(2) Fluorescence staining

The specimens were stained with fluorescent dye Hoechst 33258 (prepared with pH 7.0 Sorensen buffer at a concentration of 0.5 μg/ml) for 12 to 15 minutes. 0.5 μg/ml) for 12-15 minutes;
(3) Black light irradiation

Rinse with tap water, add 1 drop of pH 8.0 Sorensen buffer, cover with a cover slip, irradiate with a black light (20 w), and then stain with a black light. (3)Black light irradiation
(4) Warm bath

(4) Warm bath: incubate 2×SSC solution at 50～60 ℃ for 2 hours, rinse with distilled water and dry;
(5) Staining

(5) Stain with 2% Giemsa at pH 6.8 for 15 minutes, and seal transparently.
3. Method 3
(1) Specimen

The mid-stage chromosome specimens were doped with BUdR and placed under a 30-watt fluorescent lamp with a distance of 3 centimeters, and illuminated for 6 hours. under a 30-watt fluorescent lamp with a 3-cm distance between the lamps for 6 hours;
(2) Warm bath

(2) Warm bath with 2×SSC solution (60 ℃) for 15 minutes;
(3) Giemsa staining for 10 minutes, can also obtain satisfactory specimens.
In addition, there are other differentiation staining methods, but they are more complicated and not often used. Among the above differentiation staining methods, method II Method 2 is the most effective among the above differentiation staining methods.

Caveat

1. BUdR is a strong mutagen and should not be used at too high a concentration, otherwise it will produce cytotoxicity;When the concentration reaches 30 μg per ml, the number of SCE can be significantly increased;2. In method 1, the incubation time of the specimen is related to the aging time, and the aging time is related to the length of the aging time.In method 1, the incubation time of the specimen is related to the aging time, the longer the aging time is, the longer the incubation time should be.

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