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RESEARCH DIVISION Laboratory Manual

 


 

Monoclonal Antibodies

If titre is positive do one of the following 2 weeks later:

  1. Boost tail vein with 20 ug- 50 ug of Antigen in PBS and proceed with fusion on day 4. OR
  2. Boost subcutaneously with 50 ug - 100 ug of Antigen in PBS and proceed with fusion 4 days later. OR
  3. Boost 3 days in a row with 15 ug Ag in PBS and proceed with fusion on 4th day.

Cell Lines:

  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-
- 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 4C.)
- AT supplement : 50X stock , Sigma A-7422. (Store at -20C.)
- 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 37C, 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.

  1. Spin down 107 cells (106 minimum) at 1200 rpm for 5 min.
  2. Aspirate medium.
  3. Resuspend in 1 ml of ice cold freezing solution.
  4. Transfer vial to an insulated freezing box and place at -70C for at least 1 hr. (could be for a couple of days).
  5. Transfer the vial from the -70C to the liquid nitrogen tank and log the entry in the freezer log.

Thawing cells:

  1. Take vial out of liquid nitrogen tank and thaw it immediately in a 37C bath (about 1 min).
  2. 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 37C.
  3. Spin at 1200 rpm for 5 min.
  4. Aspirate medium and resuspend cells in 5 ml of medium, in a 25cm2 flask.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998