Standard Operating Procedure Form F-78 Cell Fixation IFU Authorized

BEHNIA SADEGHI 

Standard Operating Procedure FormCell Fixation

Notes

Use fresh fixative each time fixing cells

Required Reagents & Equipment (Not Supplied)

Colcemid (10 µg/ml)

Hypotonic Solution: 0.075 M KCl in H2O (formulated as 5.6 g potassium chloride per liter distilled water)

Fixative: 3:1 Methanol: Acetic Acid 15 ml conical tube

Centrifuge set to 1000 rpm for 5 minutes

Protocol

Transfer 1 ml of peripheral blood or 0.5 ml of bone marrow to a 15 ml conical tube

Optional (if trying to yield metaphase spreads): Add Colcemid (10 µl of 10 µg/ml for peripheral blood and 5 µl of 10 µl/ml for bonemarrow) and incubate for 30 minutes prior to harvest to arrest cells at metaphase. This step gives an indication about rearrangements on

hybridized probes

Pellet the cells from cell suspension, leaving some liquid so the pellet is not lost. Resuspend the cells in the remnant liquid by tapping the tube, making sure there are no clumps.

Add 10 ml of hypotonic solution drop by drop to the tube. Mix by inverting tube. Leave at 37°C for 20 minutes

Slowly add 2 ml of fixative and mix by inverting tube

Pellet the cells. Remove the supernatant, leaving some liquid to avoid loss of cells

Resuspend the cells, making sure there are no clumps

Add 10 ml of fixative, making sure there are no clumps. Mix by inverting the tube gently  Pellet the cells

Wash 2 more times with 5 ml fixative each time

Pellet and re-suspend in the required volume of fixative to drop on slides. Refer to FISH Slide Dropping Protocol for dropping details

Place remaining cells in fridge with5 5 ml of fixative for storageat ~4 °C

References

Barch MJ, Knutsen T, Spurbeck JL. The AGT Cytogenetics Laboratory Manual, Third Edition. Lippincott-Raven Philadelphia. 1991

Behnia Sadeghi
Director
Golden Genes Company