Gen5 plate reader

The spike protein level on exosomes was measured by an ELISA using precoated ELISA plates (catalog number ELV-COVID19S1; RayBiotech), according to the manufacturer’s instructions, at RT. The nucleocapsid level on exosomes was measured by an ELISA using precoated ELISA plates (Legend Max SARS-CoV2 nucleocapsid protein ELISA kit, catalog number 448007; BioLegend), according to the manufacturer’s instructions, at RT. Briefly, samples and standards were loaded onto the precoated plate and incubated for 2 to 2.5 h at RT on a shaker (200 rpm). Plates were washed and incubated with a biotin-conjugated detection antibody for an hour at RT, followed by a 45-min incubation in a streptavidin solution. After washes, plates were developed using the TMB substrate. After a 30-min incubation, the reaction was stopped by the addition of a stop solution, and the absorbance at 450 nm was recorded using a BioTek Gen5 plate reader (Agilent). For nucleocapsid, after a 10-min incubation with the TMB substrate, the absorbance was recorded at 450 nm and 570 nm. For analysis, the absorbance at 570 nm can be subtracted from the absorbance at 450 nm, and the OD can be used to build a standard curve.

The spike protein level on exosomes was measured by an ELISA using precoated ELISA plates (catalog number ELV-COVID19S1; RayBiotech), according to the manufacturer’s instructions, at RT. The nucleocapsid level on exosomes was measured by an ELISA using precoated ELISA plates (Legend Max SARS-CoV2 nucleocapsid protein ELISA kit, catalog number 448007; BioLegend), according to the manufacturer’s instructions, at RT. Briefly, samples and standards were loaded onto the precoated plate and incubated for 2 to 2.5 h at RT on a shaker (200 rpm). Plates were washed and incubated with a biotin-conjugated detection antibody for an hour at RT, followed by a 45-min incubation in a streptavidin solution. After washes, plates were developed using the TMB substrate. After a 30-min incubation, the reaction was stopped by the addition of a stop solution, and the absorbance at 450 nm was recorded using a BioTek Gen5 plate reader (Agilent). For nucleocapsid, after a 10-min incubation with the TMB substrate, the absorbance was recorded at 450 nm and 570 nm. For analysis, the absorbance at 570 nm can be subtracted from the absorbance at 450 nm, and the OD can be used to build a standard curve.

Growth inhibition effect of ESBL/AmpC E. coli strains was performed following previous described method.⁵⁹ Firstly, ESBL/AmpC E. coli strains ESBL102 and ESBL145 was chosen as hosts because of their genetic diversity. Secondly, Phages AV114, AV118 and AV125 were chosen as phage cocktail against ESBL102 whereas phages AV110, AV111, AV114 and AV125 were combined against ESBL145. Both single phages and the cocktail were evaluated for their growth inhibition ability against the strains. A single colony of each strain were inoculated in 5 mL LB media and incubated overnight at 37°C and 180 rpm. The following morning, the cultures were diluted to 1∗10ˆ8 CFU/mL and 100 μL of the culture was added to 96 wells plates (TPP). Afterwards, the phages (MOI of 10) were added to the samples and the plates were incubated in Gen5 plate reader (Agilent BioTek) for 24 hours at 37°C and OD600 values were determinate every 15 minutes. ESBL102 and ESBL145 without phages were used as negative controls. The growth inhibition assay was done in three triplicates and the standard deviations were calculated in GraphPad Prism9 (version 9.5.0).