1) Myeloma; P3X63-Ag8.653. Origin: BALB/c, non secreting, 8-azaguanine resistant, HPRT -.

2) Myeloma fox-NY. Origin: Robertsonian, 8-azaguanine resistant, HPRT -, APRT -. (mice have resistance to drug and expression of heavy chain on the same chromosome).

3) Macrophage-derived J774A.1

Maintenance of the cells:

Stock solutions:

IMDM:

Fetal Calf Serum-

Transferrin: 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 1.5E5 cells/ml in the medium described on next page. (Fresh splenic lymphocytes can be used instead if they are available). 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 recomended. (Could be aliquoted and stored at -20 C.)

Preparation of Media:

for P3X63-Ag8.653: IMDM complete to 425 ml of IMDM add: 0.5 ml 1000X transferrin; 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; transferrin; 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 3.5E5/ml.

The Macrophages optimal density is 1.5E5/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.

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