Indirect immunofluorescence (IFC) was done using standard protocol. A list of antibodies, dilutions, source and catalogue number are provided in Table S2 . Images were acquired on an inverted fluorescent microscope and analysed using NIS elements software (Nikon TS100; Melville, NY). To evaluate bacterial infectivity and intracellular S. aureus routing, cells were incubated in serum‐free EpiLife media without calcium and magnesium (Thermo/Fisher) for 2 h. Then, inactivated, fluorescently‐labelled S. aureus (Invitrogen) was added to cultures (1 × 106 particles/ml) for 4 h or overnight. After extensive washes, cells were fixed in 4% PFA and subjected for IFC detection of the intracellular structures using protein‐specific antibodies. Slides were imaged on confocal Zeiss LSM780 NLO confocal microscope and analysed using Zeiss LSM imaging software (White Plains, NY).
S aureus
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
S. aureus is a bacterial strain commonly used in laboratory settings. It is a Gram-positive, spherical-shaped bacteria that is known for its ability to cause a variety of infections in humans. The strain is often used in research and testing to evaluate the effectiveness of antimicrobial agents and other medical interventions.
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Lab products found in correlation
S aureus, by American Type Culture Collection (2 mentions)
P aeruginosa, by Thermo Fisher Scientific (2 mentions)
K pneumoniae, by Thermo Fisher Scientific (2 mentions)
Lsm 780 nlo confocal microscope, by Zeiss (1 mentions)
Epilife media, by Thermo Fisher Scientific (1 mentions)
Ts100, by Nikon (1 mentions)
Lsm imaging software, by Zeiss (1 mentions)
P aeruginosa, by American Type Culture Collection (1 mentions)
S pneumoniae, by American Type Culture Collection (1 mentions)
H influenzae, by American Type Culture Collection (1 mentions)
H influenzae, by Thermo Fisher Scientific (1 mentions)
S pneumoniae, by Thermo Fisher Scientific (1 mentions)
Staphylococcus aureus, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Facsdiva, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions)
Zymosan a, by Thermo Fisher Scientific (1 mentions)
Anti thy1.1 apc clone his51, by Thermo Fisher Scientific (1 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (1 mentions)
14 protocols using s aureus
1
Intracellular Routing of Staphylococcus aureus
2
Isolates From American Type Culture Collection
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Isolates used in this study were obtained from the American Type Culture Collection (ATCC): H. influenzae (ATCC 49766), S. aureus, (ATCC 29213), S. pneumoniae (ATCC 49619) and P. aeruginosa (ATCC 27853). All isolates were stored at -80 °C prior to inoculation onto chocolate blood agar (H. influenzae: Oxoid, Basingstoke, UK) or blood agar (S. aureus, S. pneumoniae, P. aeruginosa: Oxoid, Basingstoke, UK) and incubated at 37 °C in 5% CO2 (H. influenzae, S. pneumoniae), or in air (S. aureus, P. aeruginosa).
3
Immunoprecipitation of S. aureus-Infected Cells
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Cells were pre-incubated with DMEM for 1 h at 37°C in 6-well plates. After pre-incubation, 100 µg/mL S. aureus (Invitrogen) was added to tissue culture cells, followed by incubation for the indicated time. The cells were then washed with PBS and lysed with lysis buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 2% glycerol, and 1 mM DTT) containing protease inhibitors. The lysates were subjected to immunoprecipitation or the GST-R5BD pull-down assay as described earlier.
4
Phagocytosis Assay for E. coli and S. aureus
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SL2 cells were soaked with 1.25 μg of dsRNAs in 96 well plate for 3 days prior to phagocytosis assays. Fluorescein-labeled E. coli (K-12 strain), and Staphylococcus aureus (Wood strain, without protein A) (Invitrogen), were washed 3 times with PBS by centrifugation and sonicated for 3 times at 50 kHz for 20 s. Twenty micrograms of bacterial particles were added to each well at 4°C, the plates were centrifuged 300xg for 3 min at 4°C, and incubated in a 27°C water bath for 20 or 40 min (for E. coli and S. aureus, respectively). Cells and bacteria were removed from plates at 4°C by pipetting 300 μL of a 0.1% Trypan Blue solution in PBS pH 5.4 to quench the fluorescein from extracellular bacteria. The mount of bacteria inside live cells was quantified by flow cytometry (Ramet et al., 2002 (link)).
5
THP-1 Phagocytosis Assay with Inhibitor
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THP-1 cells were either treated with 10 µM cytochalasin D (PHZ1063, Gibco) at 37 °C and 5% CO2 for 30 min or left untreated. S. aureus, E. coli, zymosan A, dextran (25 µg/mL, 10 kDa, P10361) or transferrin particles conjugated with pHrodo red were purchased from Thermo Fisher Scientific. Particles were added to cells and cells were incubated at 37 °C and 5% CO2 for 1 h. Of note, cytochalasin D was not removed before addition of particles and the final concentration during particle uptake was 7.5 µM. After the incubation, cells were placed on ice to stop further phagocytosis. Cells were stained with anti-Thy1.1-APC (clone: HIS51, eBioScience) and DAPI (Thermo Fisher Scientific), and analyzed on BD LSRFortessa X20 (BD Biosciences). Cells were gated based on FSC/SSC, singlets, live (DAPI-) and Thy1.1+ cells using FlowJo V10.5.3 or FACS DIVA (Becton, Dickinson & Company). The mean fluorescence intensity (MFI) was determined and normalized to the MFI of non-transduced cells.
6
Investigating MEK1/2 Inhibition in Cell Culture
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The MEK1/2 inhibitor compounds PD0325901 (catalog # S1036), CI-1040 (catalog # S1020), and trametinib (catalog # S2673) were purchased from Selleck for use in in vitro cell culture experiments. PD0325901 and trametinib were used at 0.5 μM and CI-1040 was used at 10 μM. PD0325901 (catalog # PZ0162) used for in vivo experiments was from Sigma. Recombinant human M-CSF (catalog # 300-25) and murine M-CSF (catalog # 315-02) were from Peprotech. Ficoll Paque Plus (catalog # 17-1440-03) was from Cytiva. Human AB serum was from Sigma (catalog # H6914). LPS from Pseudomonas aeruginosa was from Sigma (catalog # L8643). Pam3CSK4 (catalog # tlrl-pms) and FSL1 (catalog # tlrl-fsl) were from InvivoGen (San Diego, CA). The pHrodo Red E. coli (catalog # P35361), S. aureus (catalog # A10010), and zymosan (catalog # P35364) bioparticles were from ThermoFisher Scientific. Antibodies used in these studies are listed in Supplementary Table 1
7
Opsonization of S. aureus by IVIg
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An S. aureus (Thermo Fisher Scientific, Waltham, MA, USA) vial was resuspended in 5 mL PBS. The vial was vortexed 3 × 15 s at the highest setting. The bioparticle solution was combined with IVIg, S2-IVIg or ds-IVIg in a 1:1 ratio. The mixture was allowed to rotate for 1 h at 37 °C. Subsequently, each tube was centrifuged at 1500× g for 10 min at 4 °C; the supernatants were removed and the pellet was resuspended in 1 mL PBS. The centrifugation and washing steps were repeated for a total of two washes with PBS. Finally, the washed pellet was resuspended in 200 µL of assay media.
8
Antibody Screening and Conjugation for Bacterial Detection
9
Drosophila Infection Bioassay Protocol
10
Inactivated E. coli and S. aureus Induce Nitric Oxide
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The MDM from each sample were seeded in 3 wells of a 48-well plate at a density of 2 × 10 5 cells per well in AIM V Medium containing 5 ng/mL of GM-CSF and incubated overnight. One well was assigned as the control and the other 2 wells were assigned as treatment groups and were exposed to inactivated E. coli (MOI 5, determined by titration (MOI 1, 5, 10, and 50) to induce maximum NO -using minimum MOI, the strain was isolated from a mastitic cow by a microbiologist, P. Boerlin, Ontario Veterinary College) or S. aureus (MOI 10, determined by titration (MOI 1, 5, 10, and 50) to induce maximum NO -using minimum MOI; Thermo Fisher Scientific Inc.) for 48 h. Supernatant from each well was collected and the concentration of nitric oxide (NO -) was measured with the Measure-iT High-Sensitivity Nitrite Assay Kit (Thermo Fisher Scientific Inc.). The concentration of NO -for every sample in the challenge groups was subtracted by its replicate in the control group.
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