50 ml conical tube

Low passaged (less than eight passages) B. burgdorferi strain B31 5A19 was kindly provided by Dr. Monica Embers (16 (link)). The B. burgdorferi B31 strain was grown in BSK-H medium (HiMedia Laboratories Pvt. Ltd.) and supplemented with 6% rabbit serum (Sigma-Aldrich, St. Louis, MO, USA). All culture medium was filter-sterilized by 0.2 µm filter. Cultures were incubated in sterile 50 ml conical tubes (BD Biosciences, CA, USA) in microaerophilic incubator (33°C, 5% CO₂) without antibiotics. After incubation for 7 days, 1 ml stationary-phase B. burgdorferi culture (~10⁷ spirochetes per milliliter) was transferred into a 96-well plate for evaluation of potential anti-persister activity of essential oils (see below).

RAMOS cells were resuspended in cR10 media at $5\times {10}^5$ cells/mL to a final volume of 10 mL in T25 tissue culture flasks (BD Biosciences, San Diego, CA USA) and either left untreated or treated with cyclosporine A (Sigma-Aldrich, St. Louis, MO USA) at 1 µg/mL. Pairs of untreated or CyA treated cells were then incubated in either 19% or 1% O ${}_2$ chambers for 24 h. Cells were then rapidly harvested into 50 mL conical tubes (BD Biosciences, San Diego, CA USA) containing 30 mL of ice cold PBS. Cells were pelleted and washed three times in 10 mL ice cold PBS. After the final wash, cells were resuspended in 1 mL ice cold PBS and transferred to 1.5 mL microcentrifuge tubes and pelleted. Supernatants were removed and cell pellets flash frozen in liquid nitrogen and stored at −80 °C until lysed for analysis.

Fertilized eggs of silkworms (Hu/Yo × Tsukuba/Ne) were purchased from Ehime Sansyu (Ehime, Japan). Hatched silkworms were raised in a laboratory as previously described [15 (link), 23 (link)]. Prolegs of fifth instar silkworms at day 2 after molting were cut and the dropped hemolymph was collected into ice-cold 50 mL conical tubes (Falcon) using a funnel. The collected hemolymph was supplemented with 0.05 mM phenylthiourea and centrifuged at 8,600 g at 4°C for 10 min. The centrifuged supernatant was frozen in liquid nitrogen and stored at -80°C. The frozen supernatant was melted and used as silkworm plasma.

Genome DNA extraction from formalin-fixed, paraffin-embedded (FFPE) specimens of surgically resected tissue was performed in an independent clinical laboratory (SRL, Tokyo, Japan). Genomic DNA mass was measured using a spectrophotometer (NanoDrop 2000C; Thermo Fisher Scientific, Wilmington, DE) as per the manufacturer’s recommendation.
Peripheral whole blood collected in EDTA tubes (BD Vacutainer Systems, Franklin Lakes, NJ) was centrifuged at 1500×g for 10 min at 4 °C and the plasma supernatant was transferred to 50 mL conical tubes (BD Falcon, Corning, NY) and stored at − 80 °C until use. Plasma DNA was isolated using the QIAmp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA was eluted in AVE buffer (50 μL). Approximately 40 μL of plasma DNA was concentrated to about 10 μL by SpeedVac (Thermo Fisher Scientific, Waltham, MA). DNA concentration was measured by Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA).

Bone marrow-derived MΦ (3 x 10⁶) were cultivated as described above, aliquoted into 50 mL conical tubes (Falcon), and allowed to rest for 1 hr at 37°C. Cells were then infected with CFSE-labeled O. tsutsugamushi. CFSE-labeling was performed as previously described [31 (link)]. Briefly, CFSE (Invitrogen) was mixed with bacterial stocks at a 1:1000 ratio and incubated in dark for 10 min at 4°C. The reaction was quenched by adding complete RPMI, centrifuged at 20,000 x g for 10 min, and washed twice with PBS prior to addition to MΦ cultures. At 4 hr of infection, cells were collected and divided equally for surface vs. intracellular staining as previously described [18 (link)]. Cells were stained with rat anti-Mincle mAb (MBL International), AlexaFluor594-conjugated chicken-anti-rat IgG (Molecular Probes Inc, Eugene, OR), and fixed in 2% paraformaldehyde overnight at 4°C prior to analysis. Data were collected by a BD LSRFortessa (Becton Dickinson, San Jose, CA) and analyzed by using FlowJo software version 10.7.2 (Becton Dickinson).

Fresh saliva was collected from healthy volunteers, who were required to rinse their mouths with water and ingest no food or beverage for at least 3–4 h before collection. The saliva was collected in sterile 50 mL conical tubes (BD Falcon, Houston, TX, USA) based on University of Arizona College of Medicine-Phoenix IRB-approved protocol. Saliva was diluted with TBE buffer (100 mM Tris-HCl pH8.0, 90 mM boric acid, and 1 mM EDTA) at 1:1 ratio, as described [18 (link)]. Samples were then aliquoted into sterile 1.5 mL Eppendorf tube at 200 µL each and stored at −20 °C till use.
On the day of an experiment, known amounts of either 2019-nCoV N Plasmid-RNA (Integrated DNA Technologies (IDT), Coralville, IA, USA; Lot no. 10006625) or intact, γ-irradiated SARS-CoV-2 virions (BEI, Manassas, VA, USA; Cat# NR-52287, Lot no. 70039067) were spiked into saliva samples. Saliva samples were heated at 100 °C for 5 min in a water bath in-tube [18 (link),20 (link)] or for 10 min on-cartridge. During the heating period evaluation, a time course was generated by heating saliva samples at 10 min, 5 min, 2 min, 1 min, or 0 min, respectively. The clinical SARS-CoV-2 RNA samples isolated from saliva and/or nasopharyngeal (NP) swabs were obtained from WREN Laboratory (WREN Laboratory, LLC; Branford, CT, USA) under a material transfer agreement (MTA).