Monday, 19 November 2007

Cytogenetics protocol

Culture and metaphase harvest of peripheral blood
Heparinized whole blood obtained via Vacutainer (Becton Dickinson) or syringe with preservative-free sodium heparin (25 U/ml)

Complete RPMI/10% FBS medium containing 50 microgram/ml gentamycin sulfate in place of penicillin and streptomycin.

100Xphytohemagglutinin-M (PHA; Life Technologies), reconstituted in sterile deionized water (store at 4 degree celcius)

10 microM methotrexate (optional)

1 mM thymidine (optional)

10 microgram/ml Colcemid (Life Technologies)

75 mM KCl (store < or equal 2 weeks at room temperature)

Fixative 3:1 (v/v) HPLC-grade absolute methanol/glacial acetic acid (prepare fresh)

15 ml sterile disposable conical polypropylene centrifuge tubes (do not use polystyrene)

TB syringe with 21-G needle (VWR Scientific; do not use preattached 25-G needle)


praspowt said...

All incubations are performed in a humidified 37 degree celcius, 5% CO2 incubator unless otherwise specified. All reagents and equipment coming into contact with live cells must be sterile.

1.Collect peripheral blood by venipuncture into a sodium heparin Vacutainer or a synringe with 25 U preservative-free sodium heparin per millilitre blood (do not use other anticoagulants). Initiate cultures as soon as possible (preferred) or store up to < or equal 4 days at 4 degree celcius. Ship at room temperature.

praspowt said...

2.Using a TB syringe with a 21-G needle, inoculate 0.25 ml whole blood into a sterile 15-ml polypropylene centrifuge tube containing 5 ml complete RPMI with 10% FBS and gentamycin. Use 0.2 ml whole blood for newborns < or equal 3 weeks old, and do not exceed 0.5 ml for any one tube. Add 0.05 ml of reconstituted 100 X PHA.

A single culture typically yields three to five full-slide preparations (or more if only part of the slide is used)

praspowt said...

3.a For standard samples: Incubate 2 to 4 days ( 3 days optimal) with tubes tilted at 45 degree (angle increases gas exchange and prevents dense packing).

3. b For newborns: Harvest as described either directly or following a 1- to 2-day culture (2 days optimal).

3.c For older patients: Harvest at 3 or 4 days as leukocytes from this age group do not seem to respond as quickly to PHA>

praspowt said...

4. Optional: For longer chromosomes and more mitotic cells, synchronize cells in the following manner.
The day before harvest, add 0.05 ml of 10 microlitre methotrexate (10-7 M final) to block DNA replication. Incubate 16 to 18 hr (but not longer than 18 hr).
on the following day, add 0.05 ml of 1 mM thymidine (10-5) M final) to release the methotrexate block. Incubate 4 hr (duration is critical).

praspowt said...

5. Within 3 to 4 days of culture (step3) or immediately after synchronizatio (step4), initate harvest by adding 25 microlitre of 10 microgram/ml Colcemid (0.05 microgram/ml final). Incubate 30 min. Centrifuge 8 min at 180 X g roo temparature. Discard supernatant.

praspowt said...

6. Add 6 ml of 75 mM KCl at room temperature and resuspend cells gently. Let stand 15 min at room temperature. To optimize conditions, adjusting the volume of KCl relative to the pellet volume will have more of an effect than increasing time.


praspowt said...

7. Add 10 to 12 drops fixative with aPasteur pipet and mix well. Centrifuge as in step 5.

praspowt said...

8. Remove all but 0.5 ml of the supernatant and resuspend the brown clumpy pellet in remaining supernatant gently but thorouguly by drawing it up and down with a Pasteur pipet. Avoid drawing in too great a volume as cells will stick permanently to the glass. Do not press the pipet tip against the bottom. Add 1 ml fixative and immediately mix gently. Adjust volume to 5 ml with fixative and mix thouroughly. Centrifuge as in step 5.

praspowt said...

9. Aspirate supernatant, resuspend pellet in 5 ml fixative, and centrifuge as in step 5.

10. Remove supernatant and resuspend pellet in a volume of fixative sufficient to produce a light milky suspension. Allow to stand 30 min at room temperature or overnight at 4 degree celcius (longer fixations often improve chromosome spreading)

11. Pepare slides and anlyze chromosome spreads