1. Plate cells at nearly confluent densities the day before fusion.

2. Thoroughly aspirate all media from the dish.

3. Add 50% PEG solution (pH 7.4) to dish; rock dish gently to spread.

4. After 1 min, aspirate PEG, then wash gently 3X with warm (37°C) serum free media. It is recommended to do no more than 2 or 3 dishes at a time, staggering the start times so that the first wash can be added before the minute is up for the next dish. It is critical to get the PEG off and the media on as quickly as possible when the minute is up.

e.g.: t=0 add PEG to dish 1

    t=30s add PEG to dish 2

    t=60s aspirate dish 1 and add media

    t-90s aspirate dish 2 and add media

    rinse each dish gently two more times (3 rinses total)

5. Feed cells with low mitogen media (0.5% - 2% horse serum). The purpose of this is to minimize cell proliferation. If mitosis occurs in heterokaryons, the result is nuclear fusion and chromosome loss, and you end up with cell hybrids, not heterokaryons.

6. Replace media again 4-6 h post-fusion. At this time, add 10-5 M ara C (+ 10-5 M ouabain for human x mouse heterokaryons). Replace media daily. (ara C, or cytosine arabinoside, kills dividing cells; ouabain kills human cells but at this concentration will not kill mouse cells or mouse-human heterokaryons).

7. Fusion is thought to be complete after 2 hours, although cell death from PEG treatment continues overnight.
 
 

PEG (polyethylene glycol) reagent

A. 50% polyethylene glycol 1500, in 75 mM HEPES, pH 8.0, Roche Molecular Biochemicals (formerly Boehringer-Mannheim Biochemicals) catalog number 783641. Warm to 37°C and use.

-or-

B. "BDH" polyethylene glycol 1000, catalog number 29574, BDH Chemicals, Ltd. Poole, England. Order BDH chemicals through Gallard-Schlesinger (516) 333-5600 or (800) 645-3044.

This is a solid wax-like material that must be melted and buffered before use. This is the material that was used in most if not all heterokaryon studies in the Blau laboratory published through 1990. However, there can be signicant variability in the toxicity and effectiveness of different lots, and the above reagent from Roche has also worked well. To prepare this reagent:

1. Liquify 5 g of PEG 1000 by autoclaving (10 minutes on slow exhaust) or melting in a microwave oven (on ~half-power for 1 minute).

2. Let cool in 37°C water bath until easily handled (do not allow to solidify).

3. Add 4.75 ml DME media prewarmed to 37°C (no serum) and 0.25 ml 7.5% NaHCO₃ (sodium bicarbonate, sterile tissue culture grade solution). Adjust pH by bubbling CO₂ through the solution. This can be accomplished by placing dry ice in a small side arm flask. Attach tubing to the side arm and then attach a cotton-plugged pipette to the tubing and place the pipette in the PEG solution. As CO₂ bubbles through the solution, the pH will acidify. Bubble the CO₂ until the solution is bright yellow (note that the phenol red indicator is no longer accurate in this PEG solution). Use pH indicator strips to confirm that the solution is about pH 7.4. Generally, you cannot overdo the bubbling if the right amount of sodium bicarbonate has been added. Warm solution to 37°C and use fresh.
 
 

Specific instructions for C2 heterokaryons:

C2 cells and adherent cells:

1. Grow C2 cells to near confluency (~90%).

2. Refeed cells with DME + 2% horse serum (fusion media, FM) overnight (this initiates withdrawal from the cell cycle and differentiation).

3. Trypsinize and replate C2 onto collagen coated dishes ( 4 X 10⁵ cells/ 35 mm dish; 1 X 10⁶ cells/ 60 mm dish). Feed with FM + ara C (10^-5M)

4. After one day, plate the other cell type onto the C2 cells at sufficient density to make a confluent culture. Feed with FM (no ara C).

5. Fuse cells with PEG as described above.
 
 

Fusing adherent cells and non-adherent cells. This protocol was developed using a non-adherent B-lymphoid cell line called UC and has also been used with a T-cell line (Jurkat). The adherent cell line used in this protocol is typically C2 but has also been used with fibroblasts.

1. Plate adherent cells on collagen coated dishes. Grow cells to near confluency (80%-90%).

2. Add non-adherent cells in FM. Centrifuge dish at 1200 rpm for 10 minutes to force non-adherent cells onto the layer of adherent cells. Alternatively, allow cells to settle undisturbed for 8-14 h by gravity.

3. Fuse cells with PEG as described above.
 
 
 
 

TABLE I. Suggested cell numbers for fusing UC lymphocyte cell line to C2 cells.

Dish Size (mm)	C2 cells	UC cells	Peg vol (mL)
35	2-3 x 105	0.5-1 x 107	1
60	6-8 x 105	1.5-3 x 107	2-2.5
100	1.5-2 x 106	5-8 x 107	5-6
150	4 x 106	1.5 x 108	10

A. Number of adherent cells (other than C2) varies according to cell type (shape, size). The goal is to have a nearly confluent layer of adherent cells at time of fusion. If too dense, cells will tend to "sheet off" the dish.

B. UC cells do not stick directly to plastic or collagen but do adhere to other cells. Pipet gently and always against side of dish. Some cell loss is unavoidable. If cells do not stick adequately, lectins (eg., PHA-P) may be used to improve adhesion in conjunction with cyto-centrifugation. Use the minimal effective lectin concentration and time of treatment as lectins are toxic to some cells. (They also induce physiological responses such as T-cell activation).

C. FM = DME + 2% horse serum. Use less serum (0.5%) if cells continue to grow. Cell lines that proliferate in low/no serum are problematic, as unfused cells will tend to overgrow. Ara C treatment helps to enrich for heterokaryons, but may be toxic after 2-3 days in this case. Ouabain selects against unfused human cells in human x mouse fusions.
 
 

Depending on the cell types used, it may prove necessary to adjust the proportion of PEG in order to optimize fusion. Extent of fusion correlates inversely with cell death. As little as 0.5% difference may make a dramatic difference.

Excessive cell loss or death.

Reduce PEG concentration slightly; check pH.

Rough handling. Pipet gently but don't allow dish to dry out. Add liquid at the same place and aspirate from the opposite side. Do not rock or agitate dish any more than is absolutely necessary. Do not aspirate PEG or washes to dryness--leave a little liquid to cushion the subsequent wash. Do not try to handle too many dishes at once. Only do 2-3 small dishes at once (35- or 60-mm) or one large dish at a time.

Adherent cells too dense at time of fusion. If fully confluent, you will favor homokaryon formation (see below). Often, cells will tend to detach as a sheet after PEG treatment.

Obtain and test a different lot of PEG.

Inadequate cell-cell contact. Increase density of one or both cell types; you need a considerable excess of the nonadherent cells--they should literally blanket the dish. If the adherent cell layer is too sparse, less fusion will occur. Heterokaryons containing an excess of the nonadherent cell type will be less adherent and tend to come off the dish.

Nonadherent cells form floating clumps. With UC cells, this is generally an indication that the cells are not healthy and growing well. Make sure the cultures do not contain a lot of dead cells (these cause clumps).

The "adherent" cell type does not itself adhere adequately to the dish, so the heterokaryons adhere even less well. Try alternative substrates. Note that some of these, eg. poly-L-lysine or "Cell-Tak" may in fact encourage the normally nonadherent cell type to stick. In this case, the number of nonadherent cells can probably be reduced. One may also need to be concerned with manipulating postfusion conditions to eliminate unfused cells if too many remain.

Some combinations of cell types do not fuse well, perhaps due to intrinsic differences in their plasma membranes. Obviously, if they differ significantly in their tolerance for PEG, the "optimal" conditions will be a compromise at best.