Cell culture plate

Mineral oil (Sigma) formed the oil phase for droplets in tubing (Fig. 2). For parked droplets, Mineral oil was replaced with NOVEC 7500 2 percent w/w PicoSurf (Sphere Fluidics, Cambridge, UK) and the droplets were generated into NOVEC 7500 2 percent w/w PicoSurf under a layer of Mineral oil in a cell culture plate (Eppendorf, Hamburg, Germany) (Fig. 3). For eDIBs the internal oil phase consisted of 4 milligrams per millilitre lipid solution (50 percent DPhPC and 50 percent DSPE-PEG2000; Avanti Polar Lipids, Alabama, USA) in hexadecane and silicone oil AR20 (2:1). The alginate phase consisted of 2 percent w/v low viscosity alginate with 50 milligrams per millilitre nanoparticulate calcium carbonate, adjusted to 0.1 molar ionic strength using sodium chloride. The external oil phase consisted of Mineral oil with 0.5 percent v/v glacial acetic acid.

HIV-1 infected cells maintained in complete medium were washed twice with HBSS containing Calcium/Magnesium (Thermo Fisher Scientific) and resuspended at 1 × 10⁶ cells/mL in the fresh complete medium. The cells were chilled on ice for 1 h before use. One hundred microliters of the medium containing 230 nM of CPP-HuscFvs, 10 nM of protease inhibitor, Darunavir, or 10 μM of nucleoside reverse transcriptase inhibitor, Tenofovir, were placed into appropriate wells of a 96-well Cell Culture Plate (Eppendorf AG). To each well was then added 100 μL of the infected cells and the plate was incubated at 37 °C in a CO₂ incubator for 24 h. The cells were removed from the virus-containing culture medium by centrifugation at 250× g, 4 °C for 10 min. The supernatant (180 μL) was transferred to new microcentrifuge tube and then re-centrifuged at 8000× g, 4 °C for 10 min to remove cell debris. The clear supernatant was used subsequently in three different experiments including: HIV-1 viral load assay (5 μL of the supernatant were diluted 200-fold with DPBS without Calcium/Magnesium (Thermo Fisher Scientific); HIV-1 RT enzymatic activity assay (60 μL of the supernatant); and HIV-1 virus infection assay (50 μL of the supernatant). All portions of the supernatant were kept at −80 °C until use.

The minimum inhibitory concentration (MIC) was determined by microdilution method in BM broth according to EUCAST recommendations [56 (link)]. Antibiotics were diluted with broth in 96-well plastic plates (Eppendorf Cell Culture Plates) at concentrations of 0.25–512 μg/mL. The wells were inoculated with 200 μL of bacterial culture (2–9 × 10⁶ CFUs/mL) and incubated at 37 °C under static conditions. The minimum inhibitory concentration was defined as the lowest concentration of a substance providing no bacterial growth during 24 h of incubation.
The minimum bactericidal concentration (MBC) was defined as the lowest concentration of a substance providing the reduction of viable cells by factor 1000 during 24 h of incubation. For this, 1000× culture dilutions were made from MIC-testing wells with no visible growth, inoculated into fresh broth, and incubated for 24 h. The MBC was taken as the lowest concentration of the substance providing no bacterial growth during 24 h after inoculation.