Harvesting for Replication Bands with Bromodeoxyuridine (BRDU)

 I. Purpose:

A. Provides differential staining of early and late replicating areas of the chromosomes. Allows identification of the early replicating X chromosome. Can be used for marker chromosome analysis. When Bromo deoxyuridine is added at the end of culture late replicating areas will stain pale and early replicating areas will stain dark.

II. Procedure:

A. Culture cells according to the standard procedure. For blood cultures harvest on regular day, for fibroblasts sub-culture cells 1 - 2 days before harvest.

B. Add 80 mcl Bromo deoxyuridine working solution to 10 ml cultures. Incubate cultures for 5-6 hours.

C. Add 65 mcl colcemid for final 15-30 minutes. Harvest and make slides according to standard procedure. Keep cultures and slides in dark (foil wrap tubes.)

D. Bake slides for 15 minutes at 90 - 95 C.

E. Put slides in coplin jar with Hoescht solution. Stain for 10 minutes.

F. Remove slides and rinse in dH20. Place slides flat in petri dish and flood with McIlvaines Buffer pH 7.2. Put dish under UV light box for 30 - 60 minutes.

G. Rinse slides in dH2O. Place slides flat on staining tray. Mix 3 ml Gurr buffer and 1 ml Leishman stain, put on slide, stain for 1 minute.

III. Solutions:

Bromo deoxyuridine working solution: 500 mcg/ml RPMI. Sterile filter, store in dark for 1 - 2 weeks.

Gurr Buffer: Dissolve 1 Gurr buffer tablet pH 6.8 (1 liter solution in 1 liter dH2O.

Hoechst 33258 solution: 1.0 mcg/ml PBS. Protect from light, store at 4 C for 1 week.

Leishman Stain: Dissolve 1 gram Leishman stain in 500 ml Methanol. Incubate at 37 C overnight, filter through Whatman paper qualitative grade 1.

McIlvaines Buffer pH 7.2: 6.5ml 0.1M Citrate + 43.5ml 0.2M Na2HPO4.

Colcemid working solution: 5mg/125ml dH2O.

IV. Reagents:

Bromo deoxyuridine: SIGMA cat# B 5002

Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33193-2P. Buffer tablets pH 6.8 (1 liter of solution) BPS 50 tablets/bottle.

Leishman Stain: SIGMA cat# L-6254. Harleco Leishman Stain, 25 gram/bottle. Store in dark.

Methanol: FISHER SCIENTIFIC cat# A412-500. Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free. 500 ml bottle.

Colcemid: GIBCO cat#23014-020 10mg bottle

Sodium Citrate: