Spectramax plus plate reader

Sandwich ELISAs were used at RT to measure the expression level of trimeric HIV SOSIP Env in both leaf extracts and as purified proteins. 96-well MaxiSorp plates (Nunc) were coated for 2 h in 100 ul of PBS containing 400 ng of plant-derived PGT145, purified by protein A and MEP HyperCel (Pall, 12035-208) chromatography. After blocking with 5% (w/v) nonfat dry milk in PBST, wells were washed 3 times with PBST, incubated with plant samples at different dilutions or concentrations for 2 h. Following 3 washes, wells were incubated with 400 ng of 2G12-KDEL-biotin for 1.5 h, washed and incubated with 10 ng of streptavidin (HRP) (AbCam, ab7403), washed 5 times, and developed with KPL SureBlue Reserve TMB Microwell Peroxidase Substrate (1-Component) (SeraCare, 5120-0083) for 6 min. The reactions were stopped with 0.5 M H2SO4, and absorbance at 450 nm measured using the SPECTRA max PLUS plate reader (Molecular Devices). Wells without leaf extracts and unfiltrated leaf extracts were used as controls. Purified CHO-derived BG505 SOSIP.664-His, a generous gift from Dr. John Moore, or plant-derived 3A4-CH505 SOSIP were used as a positive control.

Overnight cultures of the various streptococcal strains were diluted 1:50 into 5 ml TH+Y (containing spectinomycin for AR01+pAR107), and grown to OD₆₀₀ 0.5. The cultures were diluted 1:10⁵ and 100 μl of the diluted sample was mixed with 100 μl medium containing twice the final concentration of peptide. For overnight endpoint assays, the samples were placed in a 96-well plate and incubated stationary for 16 h at 37°C, at which point samples were resuspended using a multichannel pipette, and absorption at 600 nm was measured using a SpectraMax Plus plate reader (Molecular Devices); experiments were carried out in triplicates. For the CFU-based assays, samples were prepared in a similar manner, however they were rotated in microfuge tubes for 3 h at 37°C, and then serially diluted and plated for CFU quantification; experiments were done in duplicates. The CFU count of each peptide-treated sample was divided by that of the equivalent untreated sample to yield the final result. Data analysis was done using Prism version 5.0c (GraphPad Software).

The tissue myeloperoxidase activity assay was performed as an index of neutrophil recruitment, as previously described [26 (link)]. The test was repeated twice, with at least 5 mice from each group and each time point included from each experiment. Briefly, the skin was cut and weighed prior to homogenization in a 0.5% hexadecyltrimethylammonium bromide phosphate-buffered (pH 6.0) solution using a polytron PT1300D homogenizer (Kinematica, Lucerne, Switzerland). The homogenates were centrifuged at 14,000 rpm for 5 min at 4 °C in a microcentrifuge and five aliquots of each supernatant were transferred into 96-well plates, followed by the addition of a 3, 3′-dimethoxybenzidine and 1% hydrogen peroxide solution. Standard dilutions of pure myeloperoxidase were also tested for their activity to construct a standard curve (OD as a function of units of enzyme activity). Optical density readings at 450 nm were taken at 1 min (which corresponds to the linear portion of the enzymatic reaction) using a Spectra Max Plus plate reader using SOFTmax Pro v. 3.0 software (Molecular Devices Corp., Sunnyvale, CA, USA). Myeloperoxidase activity was expressed as units of enzyme per gram of tissue.