Monoclonal Antibodies
If titre is positive do one of the following 2 weeks later: Boost tail vein with 20 ug- 50 ug of Antigen in PBS and proceed with fusion on day 4. OR Boost subcutaneously with 50 ug - 100 ug of Antigen in PBS and proceed with fusion 4 days later. OR Boost 3 days in a row with 15 ug Ag in PBS and proceed with fusion on 4th day. Cell Lines: Myeloma; P3X63-Ag8.653
| - | Origin: BALB/c, non secreting, 8-azaguanine resistant, HPRT -. |
Myeloma fox-NY
| - | Origin: Robertsonian, 8-azaguanine resistant, HPRT -, APRT -. |
| - | (mice have resistance to drug and expression of heavy chain on the same chromosome). |
Macrophage-derived J774A.1
Maintenance of the cells: Stock solutions:
| - | IMDM: |
| - | Fetal Calf Serum- |
| - | Transferring: Iron saturated. 1000X stock = 1 mg/ml |
| - | HT supps: 50X from Sigma H0137 ( Store at -20° C ). |
| - | 2-Mercaptoethanol: 1000X stock (5 x 10 -2). (Store at 4°C.) |
| - | AT supplement : 50X stock , Sigma A-7422. (Store at -20°C.) |
| - | Kanamycin Sulfate: 100X from Gibco-BRL 600-5160AG . (Store aliquots at -20° C) |
| - | MCM: Macrophage Conditioned Medium. (used instead of feeder cells) |
Seed macrophages at a density of 1.5x105 cells/ml in the medium described on next page. Add 2.5 ug/ml LPS which induces differentiation.
Collect sup after 2-3 days, or when medium is getting too yellow. Induce 2 more times , each time with 1 ug/ml LPS and collect sup after 2 days each.
Pool the sups, filter and use as recommended. (Could be aliquoted and stored at -20C.)
Preparation of Media:
for P3X63-Ag8.653: IMDM complete to 425 ml of IMDM add:
| - | 0.5 ml 1000X transferring |
| - | 0.5 ml 1000X 2- Mercaptoethanol |
| - | 10 ml 50X HT |
| - | 5 ml 100X Kanamycin Sulfate |
| - | 75 ml FCS ( final 15 ) |
for fox-NY: IMDM or RPMI
| - | 10 FCS |
| - | 1X AT supplement |
| - | transferring |
| - | Kanamycin |
for J774A.1: same medium as the one for the P3X63-Ag8.653 ( = Ag8 ).
Growth conditions:
| - | All cell lines mentioned above grow at 37°C, 7 CO2 . |
| - | The Myelomas optimal density is 3.5x105/ml. |
The Macrophages optimal density is 1.5x105/ml. When expanding them , use a "policeman" to scrape them; this is easier to do if they are growing in petri dishes at this stage.
Freezing Hybridoma / Myeloma / macrophages.
Freezing solution: 90 FCS + 10 DMSO, ice cold.
Spin down 107 cells (106 minimum) at 1200 rpm for 5 min. Aspirate medium. Resuspend in 1 ml of ice cold freezing solution. Transfer vial to an insulated freezing box and place at -70°C for at least 1 hr. (could be for a couple of days). Transfer the vial from the -70°C to the liquid nitrogen tank and log the entry in the freezer log.
Thawing cells: Take vial out of liquid nitrogen tank and thaw it immediately in a 37°C bath (about 1 min). When there is still a small piece of ice left, dilute the cells by transferring them into a conical tube containing 10 ml of the growth medium at 37°C. Spin at 1200 rpm for 5 min. Aspirate medium and resuspend cells in 5 ml of medium, in a 25cm2 flask.
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