Media:

M-O: RPMI-1640 plus 1% of 45% D-glucose

M-10: MO plus 10% heat inactivated FCS

Hy-HAT:

RPMI-1640 - 77 ml

NCTC-135 - 10 ml

FCS (hyclone) - 10 ml

45% D-glucose (w/v) - 1 ml (0.2 micron filtered)

200 mM L-glutamine - 0.1 ml (0.2 micronfiltered)

0.1 mm Na₂SeO₃ - 0.1 ml (0.2 micron filtered)

HAT concentrate (100x) - 1 ml (0.2 micron filtered)

Gentamycin (50 mg/ml) - 0.1 ml

Hy-HT:

Replace HAT concentrate in Hy-HAT with HT concentrate (100x)

HyM-(HT or HAT):

Hy-(HT or HAT) - 98.5 ml

LPS conc. - 1.0 ml (aseptically)

Dextran sulfate conc. - 0.5 ml (0.45 micron filtered)

HAT concentrate (100x):

Hypoxanthine - 135.00 mg (H)= 1x10^-4M

Aminopterin - 1.91 mg (A)= 4x10 ^-7M

Thymidine - 38.60 mg (T)= 1.6x10^-5 M

Add (H) to approximately 60 ml of distilled water plus 1.2 ml of 1 M NaOH. Stir on magnetic stir table for 10-30 min. If not dissolved, add more 1 M NaOH in 0.1 ml volumes at 5 min. intervals until a complete solution is achieved. Next, add (T), stir until dissolved and then add (A). When all ingredients are in solution, bring the volume to 100 ml and filter sterilize through 0.2 micron filter. When stored at 4⁰ C, it is stable for several months.

HT concentrate (100x):

Same as HAT without aminopterin.

Dextran sulfate conc. (200x): (not needed if macrophages are used)

Dissolve 40 mg of dextran sulfate (17% S) MW=500,000 (Sigma), in 10.0 ml of distilled water. Filter through 0.2 micron filter and store at 4⁰ C. Stable for several weeks.

LDS concentrate (100x): (not needed if macrophages are used)

Aseptically add 20 ml of distilled water to 100 mg vial of E. coli lipopolysaccharide, W, 055:B5 (Difco) and aliquot 1 ml per tube. Store frozen at -20ð0 C. Stable for several months.

Na₂SeO₃ stock solution (1000 x):

Dissolve 1.73 mg of sodium selenite in 100 ml of distilled water. Filter sterilize through a 0.2 micron filter. Caution: Sodium selenite is a possible carcinogen.

Fusion reagent:

Polyethylene glycol-4000 (Baker) 50% :

PEG-4000 - 10 g (melted)

DMSO - 1 ml

0.15 M HEPES pH 7.5 - 9 ml

Filter sterilize with 0.45 micron filter. Store at room temperature. Stable for several months.

Hemolytic reagent:

Tris base - 0.2 g

NH₄Cl - 0.83 g

Adjust pH to 7.2 with HCl. Dissolve in 100 ml d. water. Filter sterilize with 0.2 micron filter. Store at 4⁰ C. Stable for several months.

Ethanol 70%

Sterile: three scissors and two forceps.

One 150 ml beaker containing 80-100 ml of 70% ethanol.

Two 5 ml syringes fitted with a 26 g x 1/2 inch needle and each containing 5 ml of M-10 medium.

Two 60 mm petri dishes, one with 3 ml and the other containing 10 ml of M-10 medium.

One tissue sieve (100 squares/inch) and pestle in a 100 mm petri dish. The sieve screen and pestle face should be wetted with M-10 and the petri dish contain 15 ml of this medium.

Instruments should be conveniently organized in a biological hood with a "stack" of three or four paper towels placed near the front of the hood upon which the alcohol cleansed mouse will be placed for the splenectomy. For each mouse's spleen used in the fusion, 5 ml of Tris-NH₄Cl and 15 ml of M-10 medium should be cooled in an ice bath.

The immunized mouse is sacrificed by cervical dislocation and is completely submerged in the 70% ethanol for approximately one minute. Excess alcohol is drained from the mouse and it is placed with its head to the technicians left on its back, upon the paper towels in the biological hood. The gloved technician uses the first pair of scissors to make a lateral incision between the hind legs. These appendages are pinned down with the right hand and the skin to the left of the incision grasped with the left hand. By moving the left hand towards the head and parallel to the body, the skin is peeled back completely exposing the abdomen. Using the second scissors and forceps, the visceral organs are exposed by carefully opening the abdominal musculature. The spleen is a dark red flattened and oblong shaped organ on the left side of the body situated above the kidney and adjacent to the stomach. Using the last scissors and forceps, remove the spleen and carefully trim off as much connective tissue and fat as possible. First rinse the spleen in the 60 mm petri dish containing 10 ml of M-10 medium then place in the second 60 mm petri dish containing 3 ml of M-10. With a medium-filled syringe in each hand, poke 30-50 holes in the spleen. Now perfuse the spleen using the whole 10 ml of M-10. Do not draw cells back into the syringe as they will be damaged by the small 26 ga. needle. Using sterile forceps, transfer the spleen to the tissue sieve. Pipet the cells obtained perfusion into a 15 ml capped test tube. Carefully and gently push the red spleen tissue through the sieve screen with the pestle. Connective and fatty tissues will be left on top of the screen. Pipet the medium from around the sieve and rinse cells from the screen. Remove sieve for cleaning. Carefully pipet cells from petri dish avoiding as many cell clumps as possible and put in a second test tube. Centrifuge spleen cells at 1000 rpm (PR-6000 IEC centrifuge) for 5 minutes.

Combine cell pellets and resuspend cells in 5 ml of ice cold hemolytic reagent. Incubate at room temperature for 10 minutes and add 5 ml of ice cold M-10 medium. Immediately centrifuge at 1,000 rpm for 5 minutes . The cell pellet should be erythrocyte-free and contain approximately 100 x 10⁶ cells. If the cell pellet has any red coloration indicating the presence of unlysed erythrocytes, the cells should be suspended in additional 10 ml of ice cold M-10 medium and recentrifuged. When the spleen cell pellet is free of red blood cells, combine with 100 x 10⁶ NS-1 BALB/c myeloma cells by resuspending the cells in 15 ml of M-0 medium at room temperature. Centrifuge at 1,000 rpm for 5 minutes, aspirate of the supernatant and resuspend cell pellet for a second wash in M-0 medium. Centrifuge as above and aspirate off all the supernatant to give a "dry" cell pellet.

CELL FUSION

Set a timer for 6 minutes. Quickly resuspend the cells in 0.5 ml of 50% PEG-4000 and start the timer. After 2 minutes, add 0.5 ml of M-0 medium and carefully mix. After each additional minute, add another 0.5 ml of M-0 until the end of 6 minute period. At this time, finish filling the test tube with M-0. Carefully invert to mix thus diluting the PEG.

Centrifuge at 750 rpm for 3 minutes and aspirate off the supernatant. Carefully add 10 ml of HAT medium at a rate sufficient to dislodge the pellet without dispersing the cells. Allow the fused cells to "rest" for 10 minutes then draw the cell pellet into a pipet and expel with just enough force to disperse the cells. Pipet the cells into a 75cm² tissue culture flask in a total of 50 to 60 ml of HAT medium. Incubate in a 5% CO₂ incubator at 37⁰ C for 2 days, then distribute the cells evenly into eight 96-well microtiter plates. The plates have had either 3,000-6,000 peritoneal macrophages added per well in 60-100 ml of HAT medium or the HAT medium should be supplemented with 50 mg/ml of LPs and 20 mg/ml of dextran sulfate. If the mouse's spleen cells respond well to these mitogens, the hybridomas will probably grow faster and produce more clones than with peritoneal macrophages. A drawback to using mitogen stimulation is that some hybridomas may develop a nutritional requirement for LPs or dextran sulfate.

Cultures should be feed with one drop per well ( approx. 60 ml ) of fresh medium every 3-5 days. When a well is full, one-half of the volume of the well should be removed by aspiration using a sterile pasteur pipet and a drop of fresh medium added.

 

A suitable assay to detect mouse antibodies to the antigen(s) of interest should be operational before cell fusion is attempted. Only a week or so is available from the time the cultures are producing sufficient amounts of antibodies to be assayed until the clones must be transferred to larger flasks or recloned to ascertain and maintain their monoclonality. Tests must be sensitive and require a small amount of hybridoma conditioned medium ( 100 ml or less )

 

RECLONING

When a microtiter well tests positive for the antibody of interest it is important to reclone as soon as possible to avoid the potential loss of the positive clone due to overgrowth by non-secreting cells. Recloning can be accomplished by either growing the hybridoma in soft agar or by a limiting dilution technique. I have personally better success with the latter method.

Limiting dilution recloning first requires an accurate cell count of the viable hybridoma cells suspended in the medium of the microtiter well in which the cells are growing. Next the cells are diluted so that when one drop of cell suspension is placed in each well of a 96-well microtiter plate, the average number of hybridoma cells will be 0.5, 1 and 3 cells per well. At least one plate ( 96 wells ) should be prepared for each dilution. If there is growth in 5 wells or less, the odds are greater than 95 % that the clones are monoclonal. If less than 80 % of the clones tested are positive, the hybridoma should be recloned a second time. When the dilutions listed above give to little or to much growth an appropriate adjustment to either a higher or lower dilution is required. The cloning efficiency of hybridomas can be greatly enhanced by the addition of 3,000-6,000 peritoneal macrophages per well or by including LPs and dextran sulfate in the cloning medium.

The fastest growing clones producing the most antibody are selected for clonal expansion. Several ampules of each clone chosen for expansion should be prepared for storage in liquid nitrogen using a standard cell freezing technique.

Antibody can be purified and concentrated from the hybridoma conditioned growth medium or high titer ascitic fluid can be prepared.

 

The NS-1 myeloma cell line is BALB/c origin so if the spleen cells are from any other mouse strain then ascites should be produced in the F1 cross between BALB/c and the other mouse strain. In all of the fusions I have prepared the spleen cells were from BALB/c mice and therefore this mouse strain was used to produce ascitic fluid.

The mice are first primed with an intraperitoneal injection of 0.3 to 0.5 ml of pristane. Pristane is a C₁₄ branched oily hydrocarbon which induces, in primed mice, an oil-granuloma. This environment is optimal for acceptance and growth of hybridomas and allows the antibody titer in ascitic fluid to reach levels 100 to 10,000 times greater than can be achieved in tissue culture medium.

Seven days after the mice are primed, one to ten million hybridoma cells are washed with sterile PBS and injected into each mouse. Seven to twenty one days latter the ascites is collected by inserting a 1 1/2" x 19 GA needle into the swollen peritoneum in the inguinal area and parallel to the spine. The mouse is positioned with the needle hub over a test tube and the ascitic fluid is collected by gravity flow. 1-8 ml of fluid can be obtained from each mouse.

Now the ascitic fluids allowed to clot and the cells and fibrin are removed by centrifugation. Heat inactivate at 56⁰ C for 20-25 minutes. Next, centrifuge at high speed to clarify and aliquot in 1 ml to vials and store in -70⁰ C freezer.

The mouse is prepared as for splenectomy except the abdominal musculature is left intact. A 5 cc. glass syringe is fitted with a 1 1/2" x 18 GA needle and filled with 5 ml of refrigerator cold (4-8⁰ C) 0.34 M sucrose ( dissolved in distilled water and filter sterilized through a 0.2 micron filter). A medial and enteral part of the abdominal musculature is held away from visceral organs with a sterile forceps to form a space for 3 ml of the sucrose solution to be injected. The needle is withdrawn and the abdomen massaged for about 15 seconds to insure maximum suspension of resident macrophages.

The abdominal musculature is again held with the forceps and the needle reinserted. The remaining 2 ml of sucrose in the syringe is forcefully injected into the abdominal cavity thus mixing with the cell suspension. This cell mix is then withdrawn into the syringe, care being taken not to puncture any organs with the needle while removing as much of the cell suspension as possible. The cell suspension is transferred into a sterile Teflon tube, enumerated, then capped and centrifuged at 1,200 rpm in a IEC PR-6000 centrifuge for 7 minutes. A tube of Teflon is used as macrophages will tenaciously adhere to glass and most plastic, but will not adhere to Teflon.

The cells form a lose pellet so care must be exercised when aspirating the supernatant not to disturb the pellet. 0.5 to 0.7 ml of sucrose solution can remain with the cells with no adverse effect on the hybridoma culture. 80-90 % of the resident peritoneal cells should be macrophages, while the majority of remaining cells are lymphocytes. 2-4 million cells should be recovered per mouse. The cells are now ready to be diluted in Hy-HAT medium and used as hybridoma feeder cells.

1. 96 well microtiter plate: 3,000-6,000 macroph./well

2. 24 well plate: 50,000 macroph./Well

3. 25 cm² tissue culture flask: 7x10⁵ to 1.4x10⁶ macroph.

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