4) The fusion:
a) The washed myeloma and spleen cells are pooled in 30 ml of PBS (room temp.) and spun gently at 1000 rpm for 10 min.
b) Aspirate the PBS and resuspend pellet gently by tapping the tube. Volume should be approx. 0.8 ml.
c) Set a timer.
d) 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 37 C. Some cell clumping will be evident.
e) 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).
f) Slowly add 37 C IMDM to 10 ml, without disturbing pellet. After adding, swirl the tube gently to mix and dilute the PEG. Do not disturb the cells.
g) Spin at 1000 rpm for 5 min.
h) Aspirate medium, resuspend cells by tapping. Slowly add 5 ml of 37 C IMDM-m.
i) Bring to 20 ml and add to the flask in the incubator. (If using feeder cells, add them at this point; 106 cells/ml).
j) 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.)
k) 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 peroxidase 1
- Substrate = ABTS - 100X (from Zymed 00-2001)1
- HRP buffer: 100mM Na Citrate pH 4.2( 490 mg citric acid + 720 mg Na citrate dihydrate + 50 ml H2O, pH 4.2 ).
- Hydrogen peroxide 30% ( 1000X )
- PBS, TBS
