*DPBS, Dulbecco’s phosphate-buffered saline; NA, non-applicable; ND, not done; SDS, sodium dodecyl sulfate; TCID₅₀, tissue culture infectious dose 50.
†Initial sample virus titers were as follows: liquid virus stock (1 × 10⁷ TCID₅₀/mL), infected cells (≈1 × 10⁷ TCID₅₀/mL, 1 × 10⁷ cells/mL), blood (≈10⁶ TCID₅₀/mL), tissue (≈10⁴ TCID₅₀/mg). Infected cells (5 × 10⁶ cells) were pelleted by centrifugation and lysed in test reagent (Buffer RLT; QIAGEN, Valencia, CA, USA), pelleted and resuspended in 250 μL DPBS before addition of test reagent (TRIzol; Life Technologies, Grand Island, NY, USA), or used directly in solution at the concentration and volume described in the table (fixative, heat, and SDS testing). Ebola virus–infected blood and liver tissue were collected from BALB/c mice infected with mouse-adapted Ebola virus at the height of infection, or from mock infected mice at a corresponding day.
‡Addition of ethanol: after contact time with Buffer AVL (QIAGEN, Valencia, CA, USA) or Buffer RLT, samples were transferred to a clean tube with 560 μL 100% ethanol (for AVL inactivation) or 600 μL 70% ethanol (for RLT inactivation) and allowed an additional 20 minutes contact time at 20°C.
§Although Spectra/Por Float-A-Lyzer G2 dialysis tubes (Spectrum Laboratories, Lawrenceville, GA, USA) were used for all other reagent removal by dialysis, Slide-A-Lyzer cassettes (Fisher Scientific, Pittsburgh, PA, USA) were used with TRIzol because the cassettes are more resistant to direct degradation by the reagent.
¶Tissue samples <1 cm³ were treated with a 7-day contact time, whereas samples >1 cm³ were treated with a 30-day contact time.
#Samples were boiled in an AccuBlock Digital Dry Bath (Sigma-Aldrich, St. Louis, MO, USA). All other heat treatments were carried out in a nonshaking water bath.
**4× SDS loading buffer contains 200 mmol/L Tris (pH 6.8), 4% SDS, 35% glycerol, 0.05% bromophenol blue and 20% 2-ME (added at the time of use). A combination of 250 μL 4× buffer, sample, and DPBS for a combined volume of 1 mL was tested; an appropriate volume of DPBS was substituted for 2-ME as necessary for testing of SDS as the single inactivating reagent.