Collect 5-10 cc horse blood in a heparinized vacutainer tube. Use within 24 hours.

Add 5 ml of complete RPMI 1640 medium to sterile 15 ml conical test tubes.

Invert the container with the blood several times. Add 0.3 ml of whole blood to each test tube.

Place the test tubes in a 37oC humidified incubator, propped at almost a horizontal position. If the incubator has 5% CO2, you can loosen the cap to maintain a proper pH level in the culture. If the incubator has no gas, keep the cap tightly screwed. The hepes buffer in the medium will maintain the appropriate pH level in the cells for this short term culture.

After 72 hours, add 50 ul of methotrexate to the culture to synchronize the cells. Return the tubes to the incubator. The methotrexate arrests the cells in S phase by blocking the nucleotide synthesis pathway. Therefore, the cells cannot continue in the cell cycle.

After 17 hours, centrifuge the tubes for 6 minutes at 1100 rpm. Aspirate off the supernatant and resuspend the cell pellet by tapping the bottom of the tubes several times.

Add 5 ml Hank’s Balanced Salt Solution. Centrifuge for 6 minutes at 1100 rpm.

Aspirate off the supernatant and resuspend the pellet. Add 5 ml of incomplete medium and 50 ul Brdu to the tube. Place the tube in the incubator. the Brdu releases the cells from the S stage and allows the cell cycle to continue.

After 5 1/2 hrs., add 50 ul colcemid to each tube. Place in 37oC incubator for 10-15 mins.

Centrifuge the tubes for 6 minutes at 1100 rpm. Aspirate off the supernatant and resuspend the pellet by tapping the bottom of the tube. Add 5 ml of 0.075 M KCl hypotonic solution to each tube. Set in 37oC water bath for 20 minutes. This exact time is critical.

Add 1 ml fix solution to each tube immediately after the 20 min. incubation. Screw the cap tightly and invert the tube a few times to evenly distribute the fix in the solution. Centrifuge the tubes 6 mins. at 1100 rpm.

Aspirate off the supernatant and resuspend the cell pellet. Add 1 ml fix solution to each tube. Tap the suspension vigorously to completely disperse the cells into the solution. Add 4 mls more of the fix solution and vigorously tap each tube to mix the solution.

You may now store the culture tubes in the freezer for many months, or you can proceed to drop the cells onto microscope slides.

Note: Any solutions you add to the culture prior to the fix should be pre-warmed at 37^oC.

Centrifuge the tubes containing the cells in fix for 6 mins. at 1100 rpm. Aspirate off the supernatant and resuspend the pellet. Add 5 ml fresh fix solution. Repeat 2-3 more times. This is done to get rid of excess cytoplasmic debris in the suspension. The debris will produce significant background signal if the cell suspension is not washed sufficiently.

After the last wash and centrifugation, resuspend the pellet in a few drops of fresh fix solution. The cells suspension should appear slightly cloudy. Vortex the tube for a few seconds.

Take a pre-cleaned microscope slide, and hold at an angle. Coat the slide with fix solution using a disposable pipette. Set the slide down and immediately place 1-2 drops of the cell suspension per slide. Make at least a few slides.

When the slide is dry, check your chromosomes under a phase contrast microscope to determine whether you have good chromosome spreads. Make adjustments to the concentration of the cell suspension as necessary. If the chromosome spreads are too close together, add more fix. If there are too few cells, you need to centrifuge and resuspend in the pellet in less fix.

Let the slides sit at room temperature overnight. The next day, bake the slides in a 60oC oven (dry incubator) for 3-4 hrs. The slides are now ready for FISH.

If you do not want to use the slides right away, you may store them in a slide box and place the box in a zip lock bag infused with gaseous nitrogen. Store the bag in a -20oC freezer.

	mark the target site on the slide with a diamond pen
	denature the chromosomes by immersing the slide for 5 mins in denaturing solution pre-warmed to 73oC in a water bath.
	immediately immerse the slide in cold 70% ethanol (on ice)
	wash slide 2 minutes each in 85% ethanol, 90% ethanol, 100% ethanol (dehydration series)
	let slide air dry. Make sure all the ethanol is evaporated.

D. Probe Selection and Preparation

Select one whole human chromosome specific probe, available from ONCOR or VYSIS company (e.g. chromosome #5) and follow the directions described below. You may repeat this procedure with as many different whole human chromosome specific probes as you desire, or until you have compared all 24 different human chromosomes against the horse chromosome set. You may use 3 different humans specific chromosome probes simultaneously, as long as each probe can be distinquished as a separate color.

	prepare the following while the slide is in the ethanol dehydration series
	mix the following in an eppendorf tube

	briefly vortex and microfuge for 2 seconds
	denature the probe by heating the tube in a 73oC water bath for 5 mins.
	place tube on 37oC slide warmer until ready to add to denatured chromosomes on the slide

	place slide on slide warmer (37oC - 45oC)
	apply 10 ul of probe mix (made in step 2 above) to target site on the slide
	add glass coverslip (22 x 22 mm) and seal the edges with rubber cement (use syringe)
	place slide in an enclosed container with water on the bottom. Incubate overnight (4-18 hrs.) in 37oC incubator.

	remove the rubber cement and coverslip from the slide.
	transfer the slide in sequential order in the following coplin jars for 10 mins. each. The jars are sitting in a 45oC water bath. Each jar contains 50 ml.

	briefly rinse the slide twice in PN buffer.
	counterstain the chromosomes by adding 50 ul of propidium iodide (PI) to the target site on the slide. Coverslip and let sit 5 mins.
	After a brief rinse in PBS, add about 10-30 ul of the PPD11 solution to the slide. Add a glass coverslip and view under the fluorescence or confocal microscope.

A. Culturing White Blood Cells

RPMI 1640 Complete medium

500 ml RPMI 1640 medium with Hepes buffer and glutamine

55 ml fetal calf serum

10 ml penicillin/streptomycin

5 ml L-glutamine

10 ml Phytohemagglutanin (PHA) M

6 ml heparin (6000 USP units/ml) (optional)

RPMI 1640 Incomplete medium

same as above, but leave out the PHA M and heparin

Methotrexate

1 x 10^-5 M. Prepared in Hank’s Balanced Salt Solution (HBSS)

Brdu (5’-bromo-2’-deoxyuridine)

1 x 10^-2 M. Prepared in HBSS

Colcemid

0.1 ug/ml. Purchased ready made from Life Technologies

Hypotonic solution

0.075 M KCl prepared in distilled water

Fix solution

3:1 methanol:glacial acetic acid

B. Hybridization

Denaturing solution: 70% formamide/2XSSC

35 ml formamide

5 ml 20X SSC

10 ml distilled water

50 ml total. pH to 7 with HCl

Hybridization mixture

50 ul formamide

20 ul 50% dextran sulfate

10 ul 20X SSC

Herring sperm DNA

10 ug/ul). Purchased from ONCOR, Inc., Gaithersburg, MD.

C. Wash and Detection

20X SSC

175.3 g NaCl

88.2 g sodium citrate

900 ml distilled water

bring volume to 1 liter with distilled water

50% formamide/2XSSC

75 ml formamide (straight from bottle)

15 ml 20X SSC

60 ml distilled water

150 ml total. pH to 7 with 1N HCl. Aliquot about 50 ml to each coplin jar

0.005% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate)

0.5 ml 5% CHAPS (made with autoclaved dH₂O. Stores in fridge for months)

Blocking reagent (Boehringer-Mannheim)

Prepare 1% blocking reagent solution in 100 mM maleic acid, pH 7.5

Anti-dig-FITC (Boehringer-Manneheim)

This is received as a powder (200 ug). Add 1 ml distilled water to the vial to make a stock concentration of 200 ug/ml. Make up 1:10 or 1:9 dilution using 1% blocking reagent.

PN buffer

Solution A: Na₂HPO₄:7H₂O solution (2 liters)

53.614 g Na₂HPO₄:7H₂O

distilled water up to 2 liters

Solution B: 0.33M NaH₂PO₄

39.6 g NaH₂PO₄

distilled water up to l liter

To get 0.1M NaH₂PO₄, add 230 ml distilled water to 100 ml 0.33 M NaH₂PO₄.

To make PN buffer, take 2 liters of solution A and 50-100 ml of solution B (0.1 M).

Bring pH to 8.

Add Ipegal (Sigma) to the PN buffer for a final Igepal concentration of 0.05%.

D. Reverse (R) - Banding

Propidium iodide (PI)

10 ul stock PI solution (10 mg/20 ml) in 1 ml distilled water, or purchase ready made from a company like ONCOR.

PPD 11 (p-phenylenediamine)

100 mg of PPD 11 free base (Sigma) diluted in 100 ml of nine parts glycerol to one part PBS, adjusted to pH 11 with 1 M NaOH. Store at -20^oC.

Phosphate Buffer Saline (PBS) (Dulbecco’s formula)

Calcium chloride:2H₂0 0.133 g/L

Magnesium chloride:6H₂0 0.1

potassium chloride 0.2

sodium chloride 8.0

potassium phosphate monobasic (anhydrous) - KH₂PO₄ 0.2

sodium phosphate dibasic (anhydrous) - Na₂HPO₄ 1.15

The regions of horse chromosomes that have homology to a specific human chromosome probe will appear yellow due to the hybridization of FITC (green) labeled human DNA probe to the red propidium iodide counterstained horse chromosome set. The remainder of the horse chromosomes should appear red. To see the result of human chromosome #15 hybridized to the horse chromosome complement, go to the "IMAGE" section of this web site and select "Horse Chromosomes".