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RESEARCH DIVISION Laboratory Manual

 


 

Cell Fusion and Selection

Solutions:

- IMDM
- IMDM complete + 15% MCM.
- PBS
- PEG 50% ( w/v ): PEG 4000 ( 50% ) from Boehringer Manheim 1 243 268
- Aminopterin: Sigma, A 5159 (50X )

 

  1. Prepare a T-150 flask with 170ml of IMDM complete with MCM instead of PCM (= IMDM - m ), and keep it in the incubator for fused cells.
  2. Isolation of spleen cells

    1. Sacrifice mouse by cervical dislocation. Immerse mouse in 70% Ethanol.
    2. Remove spleen ( on the left side ) and transfer into a small petri dish which contains IMDM at room temp. Clean fat from the spleen and transfer the spleen into an empty dish.
    3. Using sharp tipped forceps, one end is punctured. A curved forceps is used to hold down the intact end, and the spleen is gently rubbed towards the opened end with another set of forceps. The cells from inside the spleen will ooze out with very little damage. Stop the process when you are left with a nearly empty, transparent skin. Collect the cells by rinsing with IMDM.
    4. Transfer cell suspension to a 15ml conical tube and let the cell debris settle out (approx. 5 min.).
    5. Remove the cell suspension (without disturbing the settled cell debris) and transfer to a 50ml conical tube. Add an additional 30ml of IMDM and pellet the cells at 1200 rpm for 5 - 10 min.
    6. Aspirate the medium, resuspend pellet and wash again with 30ml of IMDM. One immunized spleen has approx. 108 cells. After this wash the cells are ready for the fusion.
  3. Myeloma cell preparation:

    It is essential that the myelomas be free of debris, rounded and refractive under phase contrast, and that they are harvested in log or late log phase growth (between 3.5 and 9 x105 cells/ml).

    1. Thaw cells 7 days before scheduled fusion. Myeloma do not grow well after being in culture for more than a few weeks.
    2. Make sure you refreeze cells for future use.
    3. We have been using a ratio of 2 spleen cells : 1 myeloma cell. However, workers have been using a ratio from 1 to 10 spleen cells per myeloma successfully. For one spleen we harvest 5x107 cells. It is advisable to do this spin at the same time that the second wash of the spleen cells is done.
  4. The fusion

    1. The washed myeloma and spleen cells are pooled in 30ml of PBS (room temp.) and spun gently at 1000 rpm for 10 min.
    2. Aspirate the PBS and resuspend pellet gently by tapping the tube. Volume should be approx. 0.8 ml.
    3. Set a timer.
    4. Add an equal volume of PEG solution, slowly, dropwise, with gentle tapping, over 1.5 min. at room temp. Then gently wiggle the tube for 1.5 min.at 37C. Some cell clumping will be evident.
    5. The suspension is spun at 1000 rpm for 3-4 min. (at this point you should see the different layers of cells with PEG on top).
    6. Slowly add 37C IMDM to 10 ml, without disturbing pellet. After adding , swirl the tube gently to mix and dilute the PEG. Do not disturb the cells.
    7. Spin at 1000 rpm for 5 min.
    8. Aspirate medium, resuspend cells by tapping. Slowly add 5 ml of 37C IMDM-m.
    9. Bring to 20 ml and add to the flask in the incubator. (If using feeder cells, add them at this point; 106 cells/ml).
    10. Add Aminopterin (2ml to 200 ml of medium). (Some workers will leave the cells at this point for 24 hours before adding Aminopterin. We add the drug immediately.)
    11. Seed the cells in 96 well microtiter dishes, 250 ul per well, 8 plates per fusion.

First clones may be seen in 7-10 days. First screen will usually start after 2 weeks, with a second and third, if necessary, a few days later.

Screening

ELISA

Materials:

- Plates - 96 well Dynatech Immulon, type 2. (Fisher 17-0221-199).
- PBS, TBS
- PBS + 0.1% Tween 20 or TBS + 0.1% Tween 20.
- Blocking solution = 2% BSA (type V ) in PBS. (Add 0.02% azide for longer storage.)
- Elisa buffer = 2% BSA + 0.1% Tween 20 in PBS ( azide optional ).
- Enzyme linked antibody = Horseradish peroxidase1
- Substrate = ABTS - 100X (from Zymed 00-2001)1
- HRP buffer: 100mM Na Citrate pH 4.2( 490 mg citric acid + 720 mg Na citrate dehydrate + 50 ml H2O, pH 4.2 ).
- Hydrogen peroxide 30% ( 1000X )

Protocol:

  1. ABSORBTION OF ANTIGEN
    1. Dilute Ag to 10 ug/ml in PBS.
    2. Add 100 ul of Ag solution to each well.
    3. Leave O.N covered with saran wrap, at 4C.
  2. BLOCKING
    1. Wash unbound Ag by inverting the plates and flicking the wells dry.
    2. Rinse by adding PBS to each well and inverting it again (use squirt bottle).
    3. Repeat the rinse twice.
    4. Add 100 ul of blocking solution to every well, leave 1 hr at room Temp or O.N at 4C.
  3. PRIMARY ANTIBODY
    1. Add the antibody to be tested:
      Sup of cells = 25 ul, mix well by pipetting up and down (10 times).serum, ascites = 1:100 and a series of 1/5 dilutions. Do dilutions in blocking solution.
    2. Leave 1 hr at room temp or O.N at 4C.
  4. SECONDARY ANTIBODY
    1. Wash unbound antibody 4 times with PBS + 0.1% Tween 20.
    2. Add 100 ul of enzyme linked antibody to all wells. Do the appropriate dilutions in the Elisa buffer. (ex: HRP is 2000X).
    3. Leave 1 hr at room temp or O.N at 4C.
  5. SUBSTRATE
    1. Dissolve substrate in water.
    2. Wash plate 4 times with PBS + 0.1% Tween 20 (use TBS instead of PBS for AP).
    3. Add 100 ul of substrate to every well.
    4. Match color development. This could take from a few seconds to 20 min.
    5. If needed, stop the reaction by adding 50 ul of 4M NaOH.
    6. Read absorption in Elisa reader at correct wavelength (for HRP system 416 nm, for AP - 405 nm).

Footnote:
  1. This is the system we have been using mostly, but there are other linked antibodies available which can be used with the same procedure
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