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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Method, Culture of Cells
Culture of Cells
• Centrifuge cells.
• Select one of the above cloning media.
• Dilute cells to 30,000 C/ml in cell cloning medium plus HAT.
• Mix well and add 100 ml/well to individual wells of a
U bottom 96 well plate.
• Incubate 37°C, 5% CO
2
.
Feeding Cells
• Optionally, replace half of the medium daily for 4 days
(not done by everyone).
• At one week, note wells that are showing signs of
metabolism by the medium turning yellow.
• Take 50 ml aliquots of supernatant for antibody screening.
• Re-feed with 150 ml complete medium plus HAT.
Cloning Cells
• Choose selected positive wells.
• Mix cells in the wells.
• Count cells.
• Dilute cells to 3 cells/900 ml (3.3 cells/ml) in cloning
medium with HAT.
• Add 100 ml/well to individual wells of a 96-well plate.
• At one week, note wells that are showing signs of
metabolism by the medium turning yellow.
• Take 50 ml aliquots of supernatant for antibody
screening.
• Re-feed with 150 ml complete medium with HAT medium.
Weaning Clones
• Clones are selected based upon immunoassays.
• Expand clone to T-25 flask.
• After growth in T-25 flask, separate out aliquot for freezing.
• Pass cells in HT medium for one passage.
• Gradually reduce the HT concentration to wean cells off HT.
Methods of Analysis
The most common method of analysis is to determine the number
of wells with clones (5 to 10,000 cells/well) that are secreting
antibody and the number that are secreting antigen specific
antibody. If low numbers of clones are obtained, it may be useful
to analyze the cells immediately after the fusion to determine if
there is a problem with the fusion itself.
A simple analysis is to use a Cytospin to place cells on a slide and
stain the cells with a Wrights or Giemsa stain. Simply count the
percent of cells with two or more nuclei.
Another method for analyzing the fusion itself is flow cytometry.
For this, cells are stained prior to the fusion with intracellular
fluorescent dyes of different color. A good method is published in
Analytical Biochemistry (1994) Vol. 216:271-275.
Producing Fusion Products Using Hybrimune
™