Tissue culture grade incubator at 37° C
10 µ g/ml Colcemid
Clinical centrifuge and tubes
0.075 M KCl
Absolute Methanol and glacial acetic acid (3:1 Mixture, prepared fresh)Slides, coverslips and permount
Alkaline solution for G-banding
Saline-Citrate for G-banding
This method works best with actively growing culture of ES cells (i.e. 1-2 day culture).
1. Add 0.1 ml of colcemid (10 micrograms/ml) to the culture flasks and incubate for an additional 2 hours.
2. The day before, passage a 70% confluent ES cell plate 1:2.
3. On the morning of karyotyping, change the medium on the plate at least 2 hours before passage and collection.
4. Trypsinize and collect ES cells into a conical tube. Centrifuge as usual and aspirate medium. Avoid allowing the pellet to dry out.
5. Gently flick the tube to resuspend the cell pellet, and add 8mL of hypotonic KCl solution to the cells. Continue to gently flick the tube during the addition of KCl to avoid clumping.
6. Incubate the tube at 37 C for 30 minutes (this may vary for each type of cell line used).
7. Add 2mL of freshly made fixative and mix by gentle inversion. 7. Centrifuge cells at 1000 rpm for 5 minutes and aspirate supernatant.
8. Using a pasteur pipette, carefully add 2mL of fixative solution dropwise, with gentle mixing to avoid clumping. Add an additional 6mL of fixative and mix by gentle inversion of the tube.
9. Centrifuge cells at 1000 rpm for 5 minutes and aspirate supernatant.
10. Repeat steps 8 & 9 three times.
11. Resuspend the pellet in 1mL of fixative (less or more according to pellet size).
1. Stain slides with freshly made Giemsa’s stain for 8 minutes.
2. Rinse in running water for 1 minute and air dry.
3. Clear cells in 2x changes of xylene and mount coverslip using Depex.
Photograph appropriate spreads and produce 8 X 10 high contrast photographs of your chromosome spreads. Cut each chromosome from the photograph and arrange the chromosomes according to size and position of the centromere.
• High quality slides need to be used. Slides should be soaked in 100% ethanol overnight and dried with lint-free tissue before use. As it is important to have slides “cold” before use, slides in ethanol bath can be stored in fridge or freezer until ready to make cell spreads.
• Some notes on KCl:- Most labs use 0.56 % KCl and some labs use 0.2% KCl + 0.2% Na citrate instead. This depends entirely on the cell types being analyzed. The time in KCl is crucial – too short and the chromosomes will be too tightly packed; too long and they will not remain in their appropriate group.
• If more detail is desired, the chromosomes can be treated with various enzymes in combination with stains to yield banding patterns on each chromosome. These techniques have become common place and will yield far more diagnostic information than giemsa stain alone (the most commonly used process). A band is an area of a chromosome which is clearly distinct from its neighboring area, but may be lighter or darker than its neighboring region. The standard methods of banding are the Q, G, R, and C banding techniques. These are defined as follows:
a. Heat hydrolysis
b. Trypsin treatment
c. Giemsa at pH 9.0
Giemsa or acridine orange
Negative bands of Q and G reversed
Heat hydrolysis in buffered salt
Pretreatment with BaOH or NaOH followed by heat and salt.
The following directions are for a G-banding:
Treat fixed and flamed slides in alkaline solution, room temperature for 30 seconds.
Rinse in saline-citrate solution for 5-10 minutes each.
Incubate in saline-citrate solution, 65° C for 60-72 hours.
Treat with 3 changes of 70% ethanol and 3 changes of 95% ethanol (3 minutes) each.
Stain in buffered Giemsa for 5 minutes.
Rinse briefly in distilled water.
Air dry and mount.