Cryogenic preservation of virus-infected cells used as immunofluorescent assay substrates

Cells infected with herpes simplex virus, respiratory syncytial virus and adenovirus were subjected to several cryogenic regimens to determine whether these procedures were detrimental to the performance and results obtained with an indirect immunofluorescence assay (IFA). Infected cells were harvested and divided into equal aliquots prior to freezing at different temperatures (-20, -70 and -165 degrees C). Cells fixed onto glass slides immediately after harvesting were used as controls. For the IFA, cells were thawed and fixed onto glass slides immediately prior to reaction with an appropriate virus-specific antibody and, subsequently, an FITC-conjugated anti-species antibody. The virus-infected cells retained their structural integrity following the experimental cryogenic regimens and were useable in the IFA. The quality of the image and the intensity of the fluorescent signal were enhanced by the incorporation of dimethyl sulfoxide into the freezing medium; cells frozen at -165 degrees C with DMSO gave the most satisfactory IFA results, although cells frozen at either -20 or -70 degrees C with DMSO were also useable. The use of cells frozen at the experimental temperatures in either phosphate buffered saline or tissue culture medium resulted in IFA substrate slides of diminished quality, often with a blurred image and distorted cellular morphology. We conclude that freezing of virus-infected cells prior to preparation for IFA is not detrimental to the performance of this procedure and the interpretation of immunological reactivity.