ClonaCell™-HY Medium D without HAT

Procedures and instruction manuals:

•  MANUAL: ClonaCell™-HY Hybridoma Cloning Kit
•  PRODUCT INFORMATION SHEET - 03810, Lot# All:29904-PIS_1_0_0.pdf

Educational resources:

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

MSDS:

•  03810-MSDS.pdf

FAQS:

• 
  
  • Q. WHY DO I GET MORE CELLS WHEN I SELECT MY FUSION IN LIQUID MEDIUM RATHER THAN IN METHYLCELLULOSE-BASED MEDIUM?
    A. Cells grown in liquid medium may appear to grow more rapidly than in methylcellulose-based medium. This is often due to the presence of a few rapidly growing clones, which multiply quickly and become abundant in liquid culture, overgrowing clones that grow more slowly. In methylcellulose cultures, the rapidly growing cells remain in close proximity to the original fusion product resulting in a large colony, preventing overgrowth of smaller, slower growing colonies.
  • 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°C 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°C.
  • 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. WHAT MYELOMA AND MOUSE STRAINS SHOULD I USE?
    A. Myeloma: There are at least two common myeloma cell lines used to generate hybridomas - SP2/0 and P3X63Ag8.683. Both of them are available from ATCC. Researchers should ensure that the myeloma line is from a reliable source and is negative for mycoplasma. Mycoplasma contamination of myeloma line can result in decreased efficiency of hybridoma formation.
    Mouse: We suggest using BALB/c splenocytes and parental myeloma cells of BALB/c for the following reasons: they are highly immune reactive, well characterized, and myeloma cells are available from the same genetic strain. However, other mouse strains will also work in ClonaCell™-HY.
  • 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.
  • Q. CAN CLONACELL™-TCS BE USED WITH ADHERENT CELL LINES?
    A. ClonaCell™ is not recommended for growth of adherent cells. If customers wish to try using ClonaCell™-TCS with their adherent line, they may wish to try a range of viscosities (diluting the ClonaCell™-TCS base medium less than recommended in the standard protocol).
  • Q. CAN CLONACELL™-TCS BE USED WITH ANY CELL LINE?
    A. A list of cell lines can be found in the manual. Other cell lines may be compatible with ClonaCell™-TCS. However it will be necessary to determine the plating and growth efficiency of the desired cells in ClonaCell™-TCS.