PNA-FISH (Q-FISH)

Metaphase Preparation:

Cultured Cells are treated with Colcemid at various time intervals depending on the cell type (lymphocytes 1/2 - 2 hrs. / fibroblasts 6 - 24 hrs.), Cells are then swollen with KCl buffer (75mM, 37o C, for approximately 30 min.) and fixed according to standard protocols with 3:1methanol/acidic acid. Fixed cells can be stored in fixative (-20oC) for at least 3 months.
For FISH the slides should be prepared freshly: Spin cells and remove old fixative. Add fresh fixative (3-5ml) and spin again. Remove half of supernatant so that cells are concentrated at ? 5x106/ml. Drop 2 spots on precleaned (Ether/Ethanol 1:1) slide. Slide should be air-dried over night.

In situ hybridization:

Rehydrate cells on slides in 1XPBS (pH=7.0-7.5) for 15 min.
Fix in 4% formaldehyde in PBS (pH=7.0-7.5) for 2 min.
Wash in 1XPBS 3x 5 min.
Treat with Pepsin 1mg/ml in 37oC for 10 min. (Pepsin should be prepared freshly in acidified water (pH=2))
Wash 2 x in PBS for 2 min.
Fix in 4% formaldehyde in PBS for 2 min.
Wash 3 x in PBS for 5 min.
Dehydrate cells in 3 steps: 5 min. 70% ethanol, 5 min. 90% ethanol, 5 min. 100% ethanol
Air dry slides
Prepare hybridization mixture:

Stock	final concentration
Formamide (ultra pure, pH=7.0-7.5)	70%
NEN in water 2.5%	0.25%
0.2M Tris	10 mM
PNA Tel Cy-3	0.5 ug/ml
PNA cen17 Cy-3	0.1 ug/ml
PNA cen16 Cy-3	0.025ug/ml
MgCl2 Buffer*	5%
dd water as diluent

Put 2 x 10 ul of hybridization mix on coverslip (22x60 mm), cover carefully with slide (upside down) and avoid air bubbles, turn the slide up.
Denature in preheated oven (80oC) for 3 min.
Place slide in plastic box and position box in a plastic beaker covered with parafilm. Hybridize for 1-2 hrs. (RT).
Remove coverslip carefully in wash solution I (70% formamide, 10mM Tris, 0.1% BSA, pH=7.0-7.5)
Wash slides 2 x 15 min. in wash solution I
Wash 3 x 5 min. in wash solution II (0.1 M Tris, 0.15 M NaCl, 0.08% Tween 20, pH=7.0-7.5)
Dehydrate cells in 3 steps: 5 min. 70% ethanol, 5 min. 90% ethanol, 5 min. 100% ethanol
Airdry slides
Put 2 x 10ul drops of vectashield containing 200ng/ml DAPI on coverslip, cover it with the slide (upside down), turn the slide up.
Keep slide in a light-protected storage box.

* MgCl2 buffer: 82 mM Na2HPO4, 9 mM citric acid, 20 mM MgCl2

Q-Fish.pdf

CO-FISH

Telomere single or dual COFISH protocol with PNA probe (Zhu, Wang & de Lange, Cell. 2004 119:355-68. Homologous recombination generates T-loop-sized deletions at human telomeres. Wang RC, Smogorzewska A, de Lange T.)

Subculture near confluent cells (1:4 to 1:8) into fresh medium containing 5’-BrdU:5’-BrdC (ratio: 3:1, reagents from Sigma) at a final concentration of 1x10-5 M and incubate for 20 hours (a little less than one cell cycle; avoid double labeling).
Four hours before harvesting, add colcemide (0.2 ug/ml for mouse cells and 0.1 ug/ml for human cells) to accumulate mitotic cells.
Take off the medium and save. Harvest cells by trypsinization, suspend in media, and spin them down in 15 ml conical tube (5 min at 1000 rpm), be gentle during harvesting, many of the dividing cells tend to lift off. Cells that have floated off during the harvest can be recaptured by spinning down the wash sup as well.
Remove supernatant completely, resuspend in 5 ml of 0.075 M KCl (prewarmed to 37oC), be gentle to prevent lysis, this step swells the cells
Incubate for 7 min at 37oC, invert the tubes to keep the cells suspended, check the cells during incubation, if they are clumping add a few drops of MeOH:Ac.Acid fixative (3:1 methanol:glacial acetic acid, prepared fresh and kept on ice) and resuspend the clumps
Spin the cells down, 5 min1000 rpm.
Decant the KCl, resuspend cells fully in the small volume of KCl that was left (by tapping)
Drop by drop add 500 ul of fixative while the cells are slowly and gently mixed on a vortex (<1000 rpm)
Add another 500 ul of fixative being less careful
Fill to 10 ml with the fixative and store at 4oC O/N or longer; cells can be kept at this stage indefinitely; when dealing with few cells, its better to spin them down from the first ml and then suspend them again in a small volume.
When ready to drop, spin the cells down (1000rpm)
Remove 9 ml of fix and resuspend cells in the 1 ml left (may vary depending on cell number)
Place a few slides in cold water; Place wet paper towels on top of a heating block set to 70 oC
Drop the resuspended cells from a couple of inches on each end of the water-wetted slide, wash the nuclei with fresh fixative (drop fixative across the slide with a bulb and Pasteur pipette; the nuclei and chromosomes should not wash off.) Place them for a minute on the humidified 70-80oC block.
Check the slides under a regular light microscope for spreading efficiency. You should see many nuclei (all of the cytoplasmic membranes should be washed away or barely visible.) Well spread metaphase chromosomes should look like small black dots at low magnification. The arms of the chromsome should be visible at higher magnifications. If the nuclei are too crowded or too sparse, you may need to dilute or concentrate your sample and drop the slide again.
Air dry the slides O/N in dark in fume hood (cover the tray with aluminum foil to keep dark and turn the light of fume hood off).
Rehydrate slides in PBS for 5 min at R.T.
Treat slides with 0.5 mg/ml RNase A (in PBS, Dnase free) for 10 min at 370C.
Stain slides with 0.5 ug/ml Hoechst 33258 (Sigma) in 2XSSC for 15 min at RT.
Expose slides to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min (equivalent to 5.4x103J/m2).
Digest the BrdU/BrdC-substituted DNA strands with 500 ul of 3 U/ul of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris-HCl, 5 mM MgCl2, and 5 mM DTT, pH 8.0) at RT for 10 min.
Denature in 70% formamide, 2xSSC at 70-800C for 1 min in vacuum oven w/o vacuum.
Dehydration in ethanol series 70%, 85%, 100%. Slides can be stored at R.T.
Follow PNA FISH protocol for FISH (Peter’s FISH protocol).
For dual COFISH: add both Tam-TelG (1:2000) and FITC-TelC (1:1000) probes together to the hybridization mix during the FISH protocol.

Solutions:
BrdU: stock concentration: 10 mM in dH2O
BrdC: stock concentration: 10 mM in dH2O

Make 1000x working stock solution of BrdU/BrdC (ratio 3:1) by mixing 7.5 mM of BrdU with 2.5 mM of BrdC.

Colcemide

Fixative

Rnase A: stock solution: 10 mg/ml in dH2O. Dilute Rnase Ato 0.5 mg/ml in PBS.

Hoechst: stock solution: 10 ug/ml in dH2O. Make a working concentration of 0.5 ug/ml in dH2O.

Exonuclease & buffer from Promega

PNA probes: FITC-TelC [FITC-OO-(CCCTAA)3] and Tamra-TelG [Tam-OO-(TTAGGG)3] (Applied Biosystems).

Flow FISH

COW THYMOCYTES

Before ordering the thymus make sure you have all these supplies:

500-1000 ml container with wide lid
400 cryo vials
labels for vials (ask Cam)
125 ml FCS
7 bottles DNase (12-14 g per bottle)
6 PBS
5 bottles Heparin (5 ml per bottle, 10.000 USPUnits/ml)
Syringes + needles
seef
5 bottles RPMI or Dulbecco’s medium
large petridish (15 cm Ø)
40 50ml Falcon tubes
funnel
formaldehyde (37-40%)

Order a thymus from Pitt Meadows Meat Farm:

Make solution (two bottles):

500 ml RPMI (or Dulbecco’s)
5% FCS (25 ml)
one bottle of Heparin
one bottle of DNase (dissolve in the solution, using a syringe)

Send this solution in a 5000-1000 ml container to the meat farm, they will send it back the next day (organise courier to bring it there and returning it the next day)

When thymus comes in:

Make 3 bottles (1500 ml) of solution (medium/FCS/Heparin/DNase).
Make 2 bottles of PBS with DNase (1 bottle DNase per 500 ml)
Take a large petridish, sterile scissors, scalpels and seef (ask Cam where to find them) and the inner part of a syringe (with black top).

Take some pieces of thymus from the container and put on petridish lid (use this as cutting plate). Pour some solution in other half of petridish. Put seef in there. Cut white stuff off thymus, cut pieces into smaller pieces, put them in seef and gently stir the tissue with the syringe-top through the seef (don’t press too hard). Pour some solution over the pieces while you are going on.
Transfer cell suspension to 50ml Falcon tubes, using a funnel. Collect about 24 tubes.
Look through the microscope if cells look okay (single cells, not too many clumps)
Spin 10 min 1500 RPM
Aspirate supernatant
Resuspend cells in 10-20 ml PBS with DNase, don’t try to resuspend the entire pellet, on bottom may be clumps, add regular PBS up to 50 ml. If there is still a pellet on bottom but you resuspended lots of cells, decant suspension into beaker.
Pool 780 ml cells in beaker, add 20 ml of 37-40% Formaldehyde. (if 800 ml is not enough, you can repeat this step later, do not take more than 800 ml at a time, you can only centrifuge 16 50ml Falcon tubes at a time)
Put on shaker for 10 min exactly.
Aliquot in 50ml Falcon tubes
Spin 5 min 1500 RPM
Aspirate supernatant, resuspend pellets
Add up to 50 ml PBS
Spin 5 min 1500 RPM
Aspirate supernatant
Resuspend pellet in 10 ml PBS per tube
Pool cells in beaker
Count cells
Dilute with PBS so you have 5x107 cells/ml
Take same volume of 80% FCS/20%DMSO
Mix the two
Aliquot in cryo vials: 1 ml cells each with repeater pipet
Freeze at -70°C

Also freeze some unfixed cells at -70°C, transfer these to -135°C.

Shipment

April 30 , 2002

CUSTOMS INVOICE

SHIPPER:

Dr. Cindy Dunbar
Head, Molecular Hematopoiesis Section
Program Director, Hematology
Building 10, Room 7C103
NHLBI, NIH
9000 Rockville Pike
Bethesda, MD 20892
301-496-1434 tel
301-496-8396 fax

CONSIGNEE:	UBC Medicine/Hematology	UBC Reference: DSAM
	Terry Fox Laboratory	Attention: Dr. Peter Lansdorp
	BC Cancer Agency	or Irma Vulto
	675 W. 10th Avenue
	Vancouver, BC, V5Z 1L3
	Canada
	Phone: (604) 675-8000 ext. 7762 or (604) 675 8135
	Fax: (604)877-0712

Shipped Via: Federal Express on Waybill # XXXXXX
1 Carton Containing: 10 samples of baboon peripheral blood

Rhesus Peripheral Blood Cells on Ice Packs (Non-Toxic; Non-Hazardous)

Total Value: $10.00 CAD.

Reason for Export: Collaborative Research

Country of Origin: U.S.A.

I hereby certify that the information given above is true and complete in every respect.

___________

Terry Fox Laboratory

British Columbia Cancer Research Centre