Biochemical and Biophysical Research Communications
Quantification of cell hybridoma yields with confocal microscopy and flow cytometry
Section snippets
Materials and methods
Preparation of rat pheochromocytoma cell line PC12 and dendritic cells (DCs). PC12 cells (ATCC CRL-1721) were grown in plastic tissue culture flasks at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO), supplemented with 10% horse serum (Sigma, St. Louis, MO), 5% fetal bovine serum (FBS, Sigma, St. Louis, MO), 2 mM l-glutamine, 1 U/ml penicillin, and 1 μg/ml streptomycin. When cells reached the desired confluence (70–80%), they were stained with vital fluorescent dyes.
Results
The aim of our work was to introduce a rapid assessment of electrofused hybridoma yields by confocal microscopy and to compare the results of electrofusion efficiency with the flow cytometric evaluation.
Discussion
Dendritic–tumor cell fusion vaccines represent a rather new possibility to improve tumor antigen presentation and provide an immunotherapeutic tool in experimental [13], [28], [29] and also in clinical [17], [19], [24], [30], [33] settings. Vaccines containing fusion hybrids are possibly more effective than other DC-based anti-tumor immunomodulatory strategies because of the presence of all tumor antigens within the context of highly expressed class I and class II major histocompatibility (MHC)
Acknowledgements
This work was supported by Grants #P3 521 0381, #P3 0310 0381, and #L2-4472-1683-03/2.06.07 of The Ministry of Education, Sciences and Sports of The Republic of Slovenia, EC Contracts: DECG QLG3-CT-2001-02004, GROWBETA QLG1-2001-02233, and the NIH Grants: FIRCA: R03-TW01293; R01 NS36665-05.
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