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One-Step Hybridoma Selection and Cloning in a 96-Well Plate
Sponsored,vendor-submitted protocol    Sponsored by STEMCELL Technologies, Inc.    Published in BioTechniques Protocol Guide 2009 (p.11) DOI: 10.2144/000113000

Abstract

STEMCELL Technologies’ ClonaCell®-HY media are optimized to support hybridoma selection and growth. Simultaneous hybridoma selection and cloning can be performed in 96-well plates. This method eliminates the need for cloning by limiting dilution, medium changes, and the harvest and expansion of large numbers of hybridoma colonies before screening, typical of other semi-solid selection approaches. In semisolid medium hybridomas develop as discrete colonies. Liquid medium is overlaid on the semi-solid medium to allow secreted antibodies to diffuse. Antibodies are screened from the liquid layer and only colonies from positive wells are plucked, resulting in considerable time and labor savings.

STEMCELL Technologies’ ClonaCell®-HY media are optimized to support hybridoma selection and growth. Simultaneous hybridoma selection and cloning can be performed in 96-well plates. This method eliminates the need for cloning by limiting dilution, medium changes, and the harvest and expansion of large numbers of hybridoma colonies before screening, typical of other semi-solid selection approaches. In semisolid medium hybridomas develop as discrete colonies. Liquid medium is overlaid on the semi-solid medium to allow secreted antibodies to diffuse. Antibodies are screened from the liquid layer and only colonies from positive wells are plucked, resulting in considerable time and labor savings.

Materials

ClonaCell®-HY Kit (STEMCELL Technologies, Cat# 03800)


Figure 1. ClonaCell®-HY 96-well procedure. (Click to enlarge)


Abbreviated Protocol

  1. Fuse splenocytes and myeloma cells according to the protocol outlined in the ClonaCell®-HY Technical Manual (www.stemcell.com) to yield between 10 and 80 million hybridoma cells. Incubate fused cells in ClonaCell®-HY Medium C for 16–24 h at 37°C.

  2. Place ClonaCell®-HY Medium D at 2–8°C to thaw overnight.

  3. On the day after fusion, vigorously shake the thawed Medium D and warm to room temperature.

  4. Determine the optimal number of cells to plate per well to arrive at 1 colony/well. (10,000–80,000 cells/well is recommended.)

  5. Resuspend the cell suspension and any desired supplements to a total volume of 10 mL in Medium C.

  6. Combine 10 mL of fused cell suspension with 90 mL Medium D. Gently invert bottle six times and allow to sit for 15 min.

  7. Use a multi-channel pipette and sterile tips (with a wide-bore), or a repeat pipette and sterile syringe, to dispense 60–80 µL Medium D into each well of a 96-well plate.

  8. Incubate plates at 37°C in a well humidified incubator for 8 days undisturbed.

  9. Remove plates and overlay 150 µL prewarmed ClonaCell®-HY Medium E onto the semi-solid medium.

  10. Incubate plates for an additional 2–4 days at 37°C.

  11. After the incubation, carefully remove 100 µL of Medium E from each well to perform antibody screening (e.g., ELISA, Western Blot, etc).

  12. The contents of positive wells should be inspected for the number of colonies it contains. A positive well containing a single colony can be resuspended with Medium E and transferred to a 24-well plate for hybridoma expansion. If a positive well contains more than one colony it may be possible to harvest the colonies separately and identify the positive clone by rescreening. If this is not possible, the hybridomas from that well will need to be recloned.

Correspondence

Address correspondence to STEMCELL Technologies, 570 West Seventh Avenue, Vancouver, BC, Canada V5Z 1B3; Tel.: (604) 877-0713, (800) 667-0322; Fax: (604) 877-0704, (800) 567-2899; http://www.stemcell.com, [email protected]

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