Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

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Abstract

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.

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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