ClonaCell®-HY Medium D

Procedures and instruction manuals:

•  MANUAL: ClonaCell® - HY Hybridoma Cloning Kit
•  PRODUCT INFORMATION SHEET - 03804, Lot# All:29517-PIS_2_0_0.pdf

Educational resources:

•  TECH BULLETIN: Techinical Note: ClonaCell®-HY 96 well plate selection and cloning (Catalog #28935)
•  VIDEO: ClonaCell® Rapid Cell Line Development
•  BROCHURE: ClonaCell® EasyPick: Rapidly Develop Cell Lines
•  BROCHURE: ClonaCell® Rapid Cell Line Development
•  VIDEO: ClonaCell™-HY Procedure

MSDS:

•  03804-MSDS.pdf

FAQS:

• 
  Q. WHY IS THERE HT (HYPOXANTHINE, THYMIDINE) IN MEDIUM E?
  
  A. Hybridomas are selected using HAT (Hypoxanthine, Aminopterin, Thymidine), which contains aminopterin. Aminopterin blocks the de novo pathway for synthesizing DNA, and the effect can persist even after the cells are removed from selection. HT provides the necessary substrates for the cells to synthesize DNA using the salvage pathway.
  
• 
  Q. IS THE SERUM IN CLONACELL^® HEAT INACTIVATED?
  
  A. Yes, all serum used in ClonaCell^® is heat inactivated.
• 
  Q. IS THERE ANY IG OR INSULIN IN THE CLONACELL^® MEDIA?
  
  A. While we don't add Ig or insulin specifically to the ClonaCell^® Media, we do add serum, which contains an undefined amount of immunoglobulins and insulin.
• 
  Q. HOW DO I THAW MEDIUM D (METHYLCELLULOSE)?
  
  A. We recommend thawing the medium overnight in a refrigerator at 4^oC and mixing well.
• 
  Q. MY MEDIUM D (METHYLCELLULOSE) APPEARS RUNNY. WHY DOES THIS HAPPEN?
  
  A. “Runny” methylcellulose could be a result of improper handling.  Over-dilution of methylcellulose, aliquoting with a pipette rather than a syringe and blunt-end needle, or insufficient mixing before use will all result in methylcellulose having altered viscosity.  Note: Methylcellulose is less viscous at RT than 37^oC.
• 
  Q. WHY DO I HAVE TO PUT MY FUSED CELLS INTO LIQUID MEDIUM OVERNIGHT? WHY CAN’T I JUST PLATE DIRECTLY INTO MEDIUM D?
  
  A. We recommend waiting up to 24 hours so that all of the fused cells can go through one cell cycle. This will ensure they have a chance to express HPRT (Hypoxanthine Guanine Phosphoribosyltransferase), the enzyme necessary to survive in the presence of aminopterin (present in Medium D). Additionally, fused cells are very fragile immediately after fusion. Waiting a day before mixing the cells with the methylcellulose will improve their survival.
• 
  Q. CAN I GROW HUMAN/RAT/T CELL HYBRIDOMAS IN CLONACELL^®-HY?
  
  A. Although we have not tried to generate human, rat or T cell hybridomas, there is no reason they should not work in ClonaCell^®-HY. The researcher needs to ensure that the cell lines used in the fusion are sensitive to HAT selection, and grow well in methylcellulose-based medium.
• 
  Q. DO I EVER NEED TO RECLONE HYBRIDOMAS GROWN WITH CLONACELL^®-HY?
  
  A. Recloning may be necessary if the number of colonies in the original dishes was very high.
• 
  Q. THERE ARE VERY FEW COLONIES GROWING IN MY MEDIUM D. WHY?
  
  A. Low numbers of colonies is generally a result of low fusion efficiency, which can have many causes. The fusion efficiency can be affected by the presence of serum during fusion, the presence of mycoplasma, low viability of cells, overexposure to PEG (polyethylene glycol), or slow growing myeloma cells prior to fusion.
• 
  Q. ONCE I PLUCK THE COLONIES AND GROW THE CELLS IN PLATES, WILL THE RESIDUAL METHYLCELLULOSE INTERFERE WITH CHARACTERIZATION? FOR EXAMPLE, WILL I HAVE PROBLEM DOING AN ELISA?
  
  A. There will likely be some residual methylcellulose contamination when colonies are plucked and transferred to the 96-well plate with the Medium E. However, the concentration of methylcellulose should be low enough that it should not show up as background.

This product has been used in:

1. Craig Dorrell et al. Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.Mol Cell Endocrinol 339 (1-2) 144-150 (June 6, 2011)
2. Masahide Yano et al. Characterization of gene use and efficacy of mouse monoclonal antibodies to Streptococcus pneumoniae serotype 8.Clin Vaccine Immunol 18 (1) 59-66 (January 2011)
3. Howard Stern et al. Development of immunohistochemistry assays to assess GALNT14 and FUT3/6 in clinical trials of dulanermin and drozitumab.Clin Cancer Res 16 (5) 1587-1596 (March 1, 2010)
4. Cassandra D Kelly-Cirino et al. Neutralizing monoclonal antibodies directed against defined linear epitopes on domain 4 of anthrax protective antigen.Infect Immun 77 (11) 4859-4867 (November 2009)
5. Shota Katori et al. Protocadherin-alpha family is required for serotonergic projections to appropriately innervate target brain areas.J Neurosci 29 (29) 9137-9147 (July 22, 2009)
6. Wei Li et al. The serine protease marapsin is expressed in stratified squamous epithelia and is up-regulated in the hyperproliferative epidermis of psoriasis and regenerating wounds.J Biol Chem 284 (1) 218-228 (January 2, 2009)
7. Kaw Yan Chua et al. Production of monoclonal antibody by DNA immunization with electroporation.Methods Mol Biol 423 509-520 (2008)
8. Lei Fang et al. Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation.J Exp Med 205 (5) 1037-1048 (May 12, 2008)
9. Kentaro Kawatsu et al. Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens.J Clin Microbiol 46 (4) 1226-1231 (April 2008)
10. Reiko Inagi et al. Establishment of a sandwich ELISA for human megsin, a kidney-specific serine protease inhibitor.Nephrol Dial Transplant 22 (11) 3311-3317 (November 2007)
11. Youjun Chen et al. Armed antibodies targeting the mucin repeats of the ovarian cancer antigen, MUC16, are highly efficacious in animal tumor models.Cancer Res 67 (10) 4924-4932 (May 15, 2007)