ClonaCell®-HY Hybridoma Kit

Procedures and instruction manuals:

•  MANUAL: ClonaCell™-HY Hybridoma Cloning Kit
•  PRODUCT INFORMATION SHEET - 03800, Lot# All:29515-PIS_2_0_0.pdf
•  PRODUCT INFORMATION SHEET - 03800, Lot# All:29518-PIS_2_0_0.pdf
•  PRODUCT INFORMATION SHEET - 03800, Lot# All:29517-PIS_2_0_0.pdf
•  PRODUCT INFORMATION SHEET - 03800, Lot# All:29516-PIS_2_0_0.pdf
•  PRODUCT INFORMATION SHEET - 03800, Lot# All:29514-PIS_2_0_0.pdf

Educational resources:

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

MSDS:

•  03801-MSDS.pdf
•  03802-MSDS.pdf
•  03803-MSDS.pdf
•  03804-MSDS.pdf
•  03805-MSDS.pdf
•  03806-MSDS.pdf

FAQS:

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  • 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.

This product has been used in:

1. 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)
2. 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)
3. Kaw Yan Chua et al. Production of monoclonal antibody by DNA immunization with electroporation.Methods Mol Biol 423 509-520 (2008)
4. 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)
5. 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)
6. 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)
7. Jon A Weidanz et al. Levels of Specific Peptide-HLA Class I Complex Predicts Tumor Cell Susceptibility to CTL Killing.J Immunol 177 (8) 5088-5097 (October 15, 2006)
8. Wayne M Yokoyama et al. Production of monoclonal antibodies.Curr Protoc Immunol Chapter 2 Unit 2.5 (September 2006)
9. Vaughan P Wittman et al. Antibody targeting to a class I MHC-peptide epitope promotes tumor cell death.J Immunol 177 (6) 4187-4195 (September 15, 2006)
10. Chunsheng Jin et al. Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice.Glycobiology 16 (4) 349-357 (April 2006)
11. Silvia C Matsumoto et al. Retinal dysfunction in patients with chronic Chagas' disease is associated to anti-Trypanosoma cruzi antibodies that cross-react with rhodopsin.FASEB J 20 (3) 550-552 (March 2006)
12. Vincent Vieillard et al. NK cytotoxicity against CD4+ T cells during HIV-1 infection: a gp41 peptide induces the expression of an NKp44 ligand.Proc Natl Acad Sci U S A 102 (31) 10981-10986 (August 2, 2005)
13. Jie Li et al. Generation of PRL-3- and PRL-1-specific monoclonal antibodies as potential diagnostic markers for cancer metastases.Clin Cancer Res 11 (6) 2195-2204 (March 15, 2005)
14. Nobuhiro Yuki et al. Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain-Barre syndrome.Proc Natl Acad Sci U S A 101 (31) 11404-11409 (August 3, 2004)
15. Hidenori Hase et al. BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex.Blood 103 (6) 2257-2265 (March 15, 2004)
16. Karen T Coffman et al. Differential EphA2 epitope display on normal versus malignant cells.Cancer Res 63 (22) 7907-7912 (November 15, 2003)
17. Q Chang et al. Structure-function relationships for human antibodies to pneumococcal capsular polysaccharide from transgenic mice with human immunoglobulin Loci.Infect Immun 70 (9) 4977-4986 (September 2002)
18. J Izard et al. Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects.J Bacteriol 183 (3) 1078-1084 (February 2001)
19. D H Song et al. Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells.J Biol Chem 275 (31) 23790-23797 (August 4, 2000)
20. E T Strovel et al. Protein phosphatase 2Calpha dephosphorylates axin and activates LEF-1-dependent transcription.J Biol Chem 275 (4) 2399-2403 (January 28, 2000)
21. Motomu Kuroki et al. Preparation of human IgG and IgM monoclonal antibodies for MK-1/Ep-CAM by using human immunoglobulin gene-transferred mouse and gene cloning of their variable regions.Anticancer Res 25 (6A) 3733-3739