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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.5/2 Appendix 1


TITLE: CLONING OF HYBRIDOMA CELL LINES BY LIMITING DILUTION


INTRODUCTION

The routine recloning of hybridoma cell lines is necessary to maintain a homogenous population of antibody secreting cells.

PROCEDURE

  1. Perform cell counts on sample to be recloned.
  2. Prepare 1ml of each line to be cloned (containing 2 x 102 viable cells) in media supplemented with 20% FCS (normally RPMI + 20% FCS 2mM Glutamine + 25mM Hepes).
  3. Dispense 100ml of the 2 x 102 viable cells/ml in wells A1-H1 of a 96 well plate which contains macrophage feeder cells.

Make serial dilutions of the cells across the columns and finally discard 100ml from column 12. See plate plan.


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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Copyright CABRI, 1998

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