Black 96 well plate

For GEF assays, the activity on His‐tagged Rabs was determined by the exchange of mant‐GDP for GTP using a PHERASTAR plate reader (Riedel et al,2017). All Rabs and TRAPP complexes were buffer exchanged into HKM (20 mM HEPES‐KOH, pH 7.4, 150 mM KOAc, 2 mM MgCl₂ and 1 mM DTT). Reactions containing 250 nM mant‐GDP‐labelled Rab alone, Rab and 200 µM GTP, or adding to the mix 10 mM EDTA, or 25 nM of the corresponding GEF, were set up in 96‐well black plates (Corning), and fluorescence decay was measured at 30°C. For the comparison between the TRAPPIII and the core effect on Rab1, 50 nM GEF was used, and the assay performed at 37°C.

Overnight culture was diluted to an OD₆₀₀ of 1.0 in 1 ml of sterile water, and this diluted culture was subsequently dispensed into individual wells of a 96-well black plates (Corning, #3915) at a volume of 100 µL per well. Fluorescence measurements were conducted using a CLARIOstar plate reader (BMG Labtech). The excitation wavelength was set at 565 nm and the emission spectrum was recorded from 590 to 615 nm. Each sample was independently replicated at least three times.

Cells (2 × 10⁴/per well) were cultivated in 96-well black plates (Corning^®, Corning, NY, USA) with 100 μL of supplemented medium (Sigma^®, St. Louis, MO, USA) and incubated for 24 h. Treatments on adherent cells were conducted at 5-20 μg/mL of samples/controls for 24 h. Then, ApoTox-Glo™ Triplex Assay (Promega^®, Madison, WI, USA) was used to determine events of viability (proliferation), cytotoxicity (necrosis), and apoptosis (caspase-3/7 activation). Additionally, apoptosis was confirmed by an ApoAlert™ DNA Fragmentation Assay (Clontech^®, Kusatsu, Shiga, J.P.). Both assays were performed according to each manufacturer’s instructions. Finally, optical density was measured in a VariosKan^® Flash (Thermo-Scientific^®, Waltham, MA, USA) [29 (link)].

The mTNFα-expressing CHO cells were seeded into 96-well black plates (CORNING) at a density of 5×10⁴ cells/well in GMEM medium. TNFα antagonists were serially diluted and incubated with the CHO cells for approximately 1 h at 4°C. The cells and TNFα antagonists were then incubated in the presence of human complement serum (Quidel) to a final concentration of 8%, for 2 h at 37°C, 5% CO₂. At the end of incubation, 50 μl of AlamarBlue^™ (Invitrogen) was added to each well, and the plate was incubated for an additional 17 h under the same conditions. Cells without TNFα antagonists and complement serum treatment served as controls. The plate was then read at 530/590 nm excitation/emissions on a SpectraMax M2^e microplate reader. For data analysis, cell death was calculated as follows: cell death (%) = (Value _control − Value _drug) / Value _control ×100. The dose-response curve was fitted with a 4-parameter model using GraphPad Prism 5 software. Rituximab was also tested against WIL2-S (CD20+) cells as a system positive control and tested against mTNFα-expressing CHO cells as a system negative control.

The protease activity assays were performed by using the EnzChek Elastase Assay Kit and EnzChek Gelatinase/Collagenase Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 10 µL of each bacterial supernatant (12 S. epidermidis (Table 1) and ATCC 12228) cultivated in 2 mL medium for 24h at 37°C, were incubated with 1 µg of DQelastin or DQgelatin (Thermo Fisher Scientific) diluted in the supplied reaction buffer (qsp 200 µL) in 96-well black plates (Corning, NY) for 20 hours. Relative fluorescent intensity was analyzed with a Fluorometer Infinite^® M200 PRO (Tecan system; excitation wavelength, 485 nm; emission wavelength, 538 nm).

Human THP1-Lucia^TM NF-κB cells were derived from the human THP-1 monocyte cell line by stable integration of an NF-κB-inducible Lucia^TM reporter construct. THP-1 Lucia NFκB reporter cells were purchased from InvivoGen (San Diego, CA, USA). THP1-Lucia^TM NF-κB cells were specifically designed for monitoring the NF-κB signal transduction pathway in a physiologically relevant cell line. The THP1-Lucia^TM NF-κB cells were grown on 96-well plate (1 × 10⁵/well) 18 h in the presence of the different concentrations FX followed by LPS (1.0 mg/L) for stimulation. For the determining of the luciferase activity, the 20 μl aliquots of cell culture media were relocated into the 96-well black plates (Corning, NY, USA) followed by QUANTI-Luc^TM assay solution (InvivoGen). Plates were measured immediately for luciferase activity with Victor 2 multiplate reader (PerkinElmer) according to the manufacturer’s instructions.

A single round infectivity assay was performed in 96-well black-plates (Corning, Kennebunk, ME, USA). Ten thousand cells/well of terminally differentiated MDMs or CHME3 or TZM-bl cells were plated 24 h prior to infection with VSV-G-pseudotyped HIV-1 at MOI 0.5 (MDMs) or MOI 0.2 (TZM-bl and CHME3 cells). Following spinoculation with virus, cells were cultured at 37 °C for 120 h (MDMs) or 48 h (TZM-bl and CHME3) and lysed, and luciferase activity was measured using the Bright-Glo luciferase substrate (Promega). Where indicated, the drugs raltegravir (10 µM), nevirapine (10 µM), or PF74 (2.5 µM) were added at indicated times after infection. Image-based analysis of infected cells was performed in an 8-well chamber glass slide using a VSV-G-pseudotyped HIVeGFP or pR9Δ virus. The percentage of infected cells was obtained by normalizing the number of infected cells expressing either the eGFP reporter or immunostained for cytoplasmic Gag to the total number of Hoechst-labeled nuclei from (n > 1000 from 4 random fields of view for each independent experiment).