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Published on: September 5, 2016
Effects of cellular fixatives on human immunodeficiency virus production
J M Cory1, F Rapp, B M Ohlsson-Wilhelm
1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey 17033.
Insights
Formaldehyde, methanol, and paraformaldehyde fixation effectively inactivate human immunodeficiency virus-1 (HIV-1) in H9 cells, reducing infectivity by over 99.99%. Dimethylsuberimidate fixation was ineffective for HIV-1 inactivation.
Area of Science:
- Virology
- Cell Biology
- Immunology
Background:
- Flow cytometry is crucial for analyzing cells, including those infected with human immunodeficiency virus-1 (HIV-1).
- Proper cell fixation is essential for safe handling of infectious samples during flow cytometry.
- Evaluating fixation effects on HIV-1 infectivity is vital for laboratory safety protocols.
Purpose of the Study:
- To assess the impact of various cell fixation methods on the infectivity of HIV-1-infected H9 cells.
- To establish guidelines for selecting appropriate fixation procedures for potentially infectious samples analyzed by flow cytometry.
Main Methods:
- HIV-1-infected H9 cells were subjected to different fixation treatments (e.g., formaldehyde, methanol, paraformaldehyde, dimethylsuberimidate).
- Fixed and unfixed infected cells were co-cultured with uninfected H9 cells to assess residual infectivity.
- Infectivity was measured by detecting cell-associated HIV-1 antigens and reverse transcriptase activity.
Main Results:
- Thirty-minute fixation with 1.85% formaldehyde, absolute methanol, methanol:acetone (1:1), or 0.5% paraformaldehyde reduced HIV-1 infectivity by over 99.99%.
- A multi-step fixation procedure using formaldehyde and ethanol also effectively reduced infectivity.
- Dimethylsuberimidate (3 µg/ml) was ineffective in reducing HIV-1 infectivity.
Conclusions:
- Standard fixation reagents like formaldehyde and methanol are highly effective in inactivating HIV-1 in H9 cells for flow cytometry.
- These findings provide critical safety guidelines for laboratories working with HIV-1-infected samples.
- Dimethylsuberimidate should not be used for inactivating HIV-1 in samples intended for flow cytometry.
Abstract:
Effects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus-1 (HIV-1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV-1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8-10 d, harvested, and evaluated for evidence of HIV-1 infection by the presence of cell-associated HIV-1 antigens and/or by the presence of particle-associated reverse transcriptase activity in cell culture supernatants. Thirty-minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV-1-infected H9 cells by greater than 99.99%. To the same degree, a multi-step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV-1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 micrograms/ml was ineffective in reducing HIV-1 infectivity.
