Cytogenetic Protocol Blood Preps

I. Preparation of materials (supply list)

1. Medium - RPMI 1640 with glutamine and hepes.  Supplement each 100 ml of medium with 1.0 ml gentamicin solution (10 mg/ml)

2. PHA - A dose curve response is necessary with each new batch of PHA to determine dosage.

3.  LPS - Dilute LPS to 750ug/ml.

4. Anticoagulant - Dilute sterile sodium heparin with supplemented medium to 500 USP units/ml (working solution). Store refrigerated.

5. Corning tubes (Fisher # 25760-16)  Prepare tubes on day of culture. Set up one tube per mouse and refrigerate until used.  Dispense into each tube:

 .95ml of supplemented RPMI 1640 medium
 0.1ml PHA to be determined (see page 4)
 0.1ml of 750ug/ml LPS (final concentration = 50 ug/ml in 1.5ml culture)
 0.15ml fetal bovine serum

6. Dispense 0.1ml working heparin solution into Fisher tubes (#14-956-3D). One tube per mouse and refrigerate until used.

II. Culture Procedure - Use aseptic technique throughout procedure until harvest.

Note: The ideal age to culture is at 7-10 weeks and females tend to provide the best cultures. Cells from mice as young as 5 weeks and as old as 2 years have been cultured with this method but the success rate and number of metaphase spreads per slide is usually greatly reduced.  

1. Obtain blood from the retro-orbital sinus using heparinized micro- hematocrit tubes (Fisher 02-668-66). Obtain 2 tubes per mouse. Inoculate blood immediately into 0.1 ml of diluted heparin (500 USP units/ml). Cap tubes tightly and swirl gently to mix blood and heparin.  [Those unskilled in this procedure may obtain blood from the tail vein. (method on page 3)].

2. Blood should be held at room temperature until inoculation. Do not hold blood for more than 2 hours.

a. Add 0.2 ml of blood and heparin mixture to each prepared culture tube.
b. Cap tubes tightly. Swirl tubes gently to mix cells with medium.

3. Incubation period

Incubate cultures at 37°C in a shaking water bath. Set shaking speed so that the basket moves back and forth about 32-35 times per minute. Place tubes at an angle in the rack and shake 3 times daily.

[An oven may also be used to incubate cultures. The rack of tubes is tilted at an angle by resting it on two 1/2 to 5/8 inch high petri dishes to allow maximum surface area  exposure.  Only a simple oven is required provided a constant temperature is maintained.  Shake tubes 3 times daily to resuspend cells].

III. Harvest

1. Colchicine treatment

a. At 41-43 hrs  add to each culture  0.15 ml of 50 ug/ml  colchicine solution.
b. Incubate tubes 15-20 min. longer.

2. Cell harvest

a. Transfer the culture for each mouse to a 5-ml conical  centrifuge tube (Kimax) and centrifuge at setting #4 on a clinical  bench-top centrifuge (approx. 400 x g) for 10 min.

b. Remove supernatant and gently add 2-3 ml of warm (37°C) 0.56% KCl  (potassium chloride).  Gently suspend cells by pipetting. Return  tubes to incubator for 15 min. Tubes may stand upright now. Centrifuge at setting #5 (approx. 500 x g).

c. Remove supernatant without disrupting pellet. Caution - do not  remove buffy coat on top of pellet. Gently add 3-4 mls fixative  down side of tube. Then pipet gently but rapidly and immediately to prevent cell clumping. Fixative = methanol:glacial acetic acid, 3:1.

d. Stopper tubes and allow to sit for at least 30 min. Refrigerate if tubes are to be held more than  30 min. The procedure may be interrupted at this point for 1-2 hours.

e. After 30 min. centrifuge cells at setting #4, and resuspend in fresh fixative. Repeat this step twice more. Cells may be left refrigerated for several days once this step is completed. Cells must be washed at least twice before leaving overnight.

f. Centrifuge once more and resuspend cells in about 1/3 to 1/2 ml of  fixative. Slides are prepared from this cell suspension by placing  the entire sample on one slide. If slides are not  made immediately, save this last wash until just prior to slide making.

IV. Slide making

a. Immerse pre-cleaned slides in fixative at least 15 min. prior to use. Wipe slides dry with a kim wipe or other lint-free tissue. Slides must be scupulously clean.

b. Drop small drops of cell suspension onto slide surface.

c. Allow drop to spread over slide surface. If too much sample has been used, it will bubble at slide edges.

d. As soon as the drop begins to contract and Newton's rings are visible (rainbow around edge of drop), blow on the slide surface to accelerate drying. Slides may also be dried by using tubing hooked to an air supply or by lightly spraying with  Dust-off Plus.

e. The "bomb" method (dropping one very small drop of fixative onto the cell suspension once it has started to dry) may be used to increase the spreading of the chromosomes. When using this method keep slide in a flat position when adding the fixative. Do not blow on slide and dry it thoroughly before adding more suspension. 

f. Repeat b through d until all the sample is on the slide. Monitor cell concentration with microscope.

Collecting blood by tail vein method

[Allow extra time for this method and collect the blood in 0.05 ml of diluted heparin (500 USP units/ml)].

a. Prewarm mouse under desk lamp in large jar 1-2 min. The mouse is  warm enough when it rubs its nose or shows excessive activity. Caution - overheated mice can go into shock.

b. Place mouse in restraint or have someone hold it so tail is free.

c. Wash the tail with 70% ethanol. Wipe twice with clean Kim wipe.

d. Cut gently across vein on side of tail about 1 inch from base of tail with a razor blade wiped with 70% alcohol.

e. Allow first 1-2 drops of blood to drop onto clean towel while getting bleeding tube.

f. Let blood run down side of tube. Be careful not to touch tail to mouth of tube. Collect at least 0.15 ml (5 drops) if possible.  This is enough for a 1.5-ml culture.

Lymphocyte growth in cultures depends upon many variables including the response to the mitogen used. PHA is used to stimulate T cell growth and LPS is used to stimulate the B cells.  We assay the mitotic index with each new batch of PHA although, in recent years, we have found that  7.5ug/ml gives the best result regardless of the mitogenic units marked on the vial. The mitotic index varies between strains because response to PHA is influenced by genetic background (Heiniger et al ).  Because of the interstrain variabilty, a dose response curve needs to be determined for each strain to be cultured.  Pool blood from each strain and prepare 2 tubes for each concentration. We suggest using a C57BL/6J  as a control  because of their good response to PHA.

PHA Dose Response

Final concentrations / 1.5ml culture:
6ug / ml
7ug / ml
8ug / ml
9ug / ml

2mg PHA / 2ml medium = 1mg/ml

PHA

Medium

Total Volume

Concentration

0.2ml

3.1 ml = 60 ug/ml

3.3ml

6 ug/ml

0.23ml

3.07ml = 70 ug/ml

"

7 ug/ml

0.265ml

3.04ml = 80 ug/ml

"

8 ug/ml

0.3ml

3.0ml = 90 ug/ml

"

9 ug/ml

Set up 2 tubes for each concentration

Add 0.15ml PHA (each dilution) to each culture
Medium 0.95ml
LPS 0.1ml
FBS 0.15ml
Heparin 0.05ml
Blood 0.1ml
Total Volume = 1.5ml