All A. baumannii reference strains and clinical isolates were cultured at 37°C in LB broth media for 16h before subsequent experiments. For IC50 determination, bacterial cells corresponding to OD600nm=0.1 were transferred to a 96 well flat bottom plate (Greiner, Austria) containing varying concentrations of HDC1 (0.1; 1; 10; 25; 50; 75; 100 and 1000 µM) in LB broth media with 1% DMSO. For determination of the activity of HDC1 on the 43 clinical A. baumannii isolates, bacterial cells corresponding to OD600nm=0.1 were transferred to a 96 well flat bottom plate (Greiner, Austria) containing 100 µM of HDC1 in LB broth media with 1% DMSO. For both experiments, the positive control included bacterial cells corresponding to OD600nm=0.1 of the used strains in LB broth media with 1% DMSO. The negative control included both LB broth media and LB broth media supplemented with 1% DMSO. Collection of data was done using the Cytation 1 (BioTek, United States). The OD600 absorbance of all bacterial cultures was measured every 30 min for 24h at a temperature of 37°C and agitation of 355 cpm (cycles per minute). In all analysis, growth inhibition is defined as decreasing absorbance values over time compared to the positive control. The data were measured in biological triplicate.
Flat bottom 96 well plate
Manufactured by Greiner
Sourced in Austria, Germany, United States
The Flat-bottom 96-well plate is a laboratory equipment used for various experimental purposes. It features a flat-bottom design with 96 individual wells, providing a standardized format for conducting assays, cell culture, and other applications that require a high-throughput approach.
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Lab products found in correlation
Infinite 200 pro, by Tecan (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Synergy mx microplate reader, by Agilent Technologies (2 mentions)
Incucyte zoom time lapse microscopy system, by Sartorius (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Bio layer interferometry instrument, by Sartorius (2 mentions)
Anti higg fc capture ahc biosensors, by Sartorius (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Oxoplate op96c, by PreSens (2 mentions)
Acetic acid, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
M1000 pro, by Tecan (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
Cell counting kit 8 reagent, by Dojindo Laboratories (1 mentions)
Orca flash 4.0 v3 camera, by Hamamatsu Photonics (1 mentions)
Cell observer microscope system, by Zeiss (1 mentions)
96 well flat bottom cell culture plate, by Greiner (1 mentions)
Ldh cytotoxicity detection kit, by Takara Bio (1 mentions)
77 protocols using flat bottom 96 well plate
1
Determination of HDC1 Antibiotic Efficacy
2
Sulforhodamine B Chemosensitivity Assay
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The sulforhodamine B (SRB) chemosensitivity assay was performed as described earlier [12 (link)] in 96-well flat-bottom plates (Greiner Bio-One). Briefly, after plating in DMEM, cells were given 24 hr to reattach to the plates and resume growth. Hereafter, the appropriate drug was added to the wells. Next, the cells were incubated at 37°C for 48 hr. Cells were then fixed with cold trichloroacetic acid (6% final concentration; Merck, Darmstadt, Germany), and incubated for at least 1 hr at 4°C. Subsequently, cells were washed with water and dried at room temperature. Fixed cells were stained with SRB dissolved in 1% acetic acid for 15 min at room temperature followed by a 1% acetic acid (Merck) wash. 10 mM Tris (Merck) base solution was added to the wells and optical density (OD) of the SRB staining was measured with a Tecan4 plate reader (Tecan, Männedorf, Switzerland) at 540 nm.
3
Fluorimetric Analysis of CIPK Proteins
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Fluorimetric analyses were performed in yeast as previously described [15 (link)]. In brief, CIPK15, CIPK19, and CIPK15m (K41N, an inactive form) were introduced into yeast expressing AmTryoshka1;3 LS-F138I. Vector only was used as the control. Cells were analyzed in 96-well, flat-bottom plates (Greiner Bio-One, Germany). Steady-state fluorescence was recorded using a fluorescence microplate reader (Infinite, M1000 pro, Tecan, Switzerland) in bottom-reading mode using 7.5 nm bandwidth and a gain of 100. The fluorescence emission spectra (λexc 440 or 485 nm; λem 510 or 570 nm) were background-subtracted using yeast cells expressing a non-florescent vector control.
4
Kinetic Assay for 1-Naphthyl Acetate Hydrolysis
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The hydrolysis of 1-naphthyl acetate (1-NA) was measured spectrophotometrically by monitoring the release of 1-naphthol at 320 nm [5 (link)] in a Synergy MX temperature regulated plate reader (BioTek) at the desired temperature. Assays were performed in 96-well flat-bottom plates (Greiner) in a final volume of 100 µL. The well contained sodium phosphate buffer (50 mM, pH 7.5), NaCl (100 mM) and 1-naphthyl acetate (0.5 mM). The reaction was initiated by adding purified protein to a final concentration of 10 µg/mL. Residual esterase activity assays were performed by incubating the enzymes in sodium phosphate buffer (50 mM, pH 7.5) containing NaCl (100 mM) in a PCR thermocycler at the desired temperature. Aliquots were taken at various incubation times (0–90 min) and assayed for 1-naphthyl acetate hydrolysis at 40 °C. A calibration curve was used to calculate the concentration of 1-naphthol released in the reaction. Specific activity was defined as the amount of protein (mg) required to form 1 µmol of 1-naphthol, per min. Measurements were performed as technical triplicates.
5
Cell Viability Assay for HT-29 Cells
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HT-29 cells (2.5 × 104 cells/well) were seeded into a 96-well flat bottom plates (Greiner) for 48 h. The cells were incubated with various concentrations of each probe for 24 h at 37 °C under a 5% CO2 atmosphere. The medium was removed, and the cells were washed gently with McCoy’s 5A medium without phenol red. Cell Counting Kit-8 reagent (CCK-8, Dojindo) was added to each well, and incubation was continued for 1 h58 (link). The absorbance at 450 nm of each well referenced at 650 nm was recorded using a microplate reader (Infinite 200 PRO, Tecan). Cell viability (% of control) was evaluated as (Asample − Ablank)/(Acontrol − Ablank) × 100, where Asample is the absorbance of cells exposed to the probe, Acontrol is the absorbance of cells without probe, and Ablank is the absorbance of wells without cells.
6
Optimized Cell Imaging and Analysis
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Cells were imaged using a CellObserver microscope system (Carl Zeiss) equipped with a 20×/0.8 PlanApo Phase 2 lens, a Hamamatsu ORCA-Flash4.0 v3 camera, a temperature controlled XL-chamber, a temperature, humidity and CO2 controlled stage incubator, a motorized coded X,Y-stage, a Definite Focus system and a HXP120 Metal-Halide illumination unit. Visual inspection of cells prepared for flow cytometry was conducted using 96-well flat-bottom plates (Greiner Bio-One, Kremsmünster, AUT).
7
Mitochondrial Membrane Potential Assay
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HT-29 cells (3.0 × 104 cells/well) were seeded into a 96-well flat bottom plates (Greiner) for 48 h. The cells were stained with Ir-1 or Ir-2 (1 μM, 2 h). The medium was removed, and the cells were washed gently with McCoy’s 5A medium without phenol red. 5,5′,6,6′-tetrachloro-1,1′-3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Thermo Fisher Scientific) (5 μM) was added to each well, and incubation was continued for 30 min. The medium was removed, and the cells were washed gently with McCoy’s 5A medium without phenol red. The emission intensities of JC-1 were measured using a microplate reader (Infinite 200 PRO, Tecan) equipped with a gas control module (GCM, Tecan). The excitation wavelength was 488 nm, and the monitor wavelength was 595 nm for Red emission (aggregate) and 540 nm for green emission (monomer).
8
Lactate Dehydrogenase Cytotoxicity Assay
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The LDH assay was performed using a commercially available LDH Cytotoxicity Detection Kit (Takara, Göteborg, Sweden) as per the manufacturer’s instructions, and as described by [15 (link)]. Briefly, varying numbers of effector cells from fish treated as described in Table 1 were added to a constant number of non-infected or SAV infected MHC-I matched or MHC-I mismatched target cells (E:T). Confirmation that the target cells had a matched or mismatched MHC-I molecule with the C25 clonal fish used in these in vivo experiments was done by RT-qPCR applying primers that specifically amplify the Onmy-UBA*501 gene as described previously [22 (link)]. The effector and target cells were incubated together at 15 °C for 4 h in 96-well flat bottom cell culture plates (Greiner, Kremsmünster, Austria), in triplicate. After the incubation period, the plates were centrifuged (10 min, 250× g, 4 °C) and 100 µL of supernatant was removed and added to new 96-well flat bottom plates (Greiner). 100 µL of cytotoxicity assay substrate was then added and the plates were left to develop at room temperature for 30 min. Plate absorbance was read in a Spectra max Plus (Analytical Technologies) plate reader at 490 nm (ref 655 nm) using Softmax Pro 5.3 software. A detailed description of background controls and the formula used to calculate cytotoxicity can be found in Appendix A .
9
Transfection of HEK-293 Cells with NOD2
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Transfection of HEK-293 cells with human NOD2 was performed as previously described by Laayouni et al.49 (link) and culture conditions are described as supplementary material. Non-transfected or NOD2-transfected HEK-293 cells (1 × 106 cells) were added to 96-well flat-bottom plates (Greiner) in the presence of either E. coli lipopolysaccharide (LPS, O111:B4, 10 ng/mL; Sigma), further purified based on50 , muramyl dipeptide (MDP, 10 μg/mL; Sigma), lysates or promastigotes (2 × 106 parasites) of L. braziliensis or L. amazonensis in a final volume of 200 μL. After 24 h of incubation, at 37 °C and 5% CO2, supernatants were collected and stored at −20 °C until analysis of IL-8 production.
10
Isolation and Proliferation Assay for Chicken PBMCs and Splenocytes
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PBMCs and splenocytes were isolated using Ficoll-paque plus (GE Healthcare, PA). Briefly, blood was diluted in PBS (1:1 ratio) and an equal volume of Ficoll-paque plus solution was added and the mixture was then centrifuged at 450xg for 25 min, 20°C. Lymphocytes at the interface were collected, washed twice in PBS and suspended in E-RPMI medium. Splenocytes were isolated by teasing splenic tissue through a cell strainer using PBS. Splenocytes were then mixed with an equal volume of Ficoll-paque plus solution and centrifuged at 450 xg for 30 min, 4°C. Splenocytes at the interface were collected, washed twice with PBS and resuspended in E-RPMI medium.
For the cell proliferation assay, PBMCs and splenocytes (1x106 cells/well in 100 µL) were suspended in E-RPMI medium and seeded in triplicate wells of 96 well flat-bottom plates (Greiner bio-one, NC). Cells were treated with OMPs (5 µg/mL in 100 µL) in E-RPMI medium and incubated for 72 h at 39°C, 5% CO2. After incubation, 20 µL of MTS+PMS solution was added and incubated for 4 h at 37°C, 5% CO2. The OD value was taken at 490 nm by the ELISA plate reader. The stimulation index (SI) was calculated by dividing OD value of stimulated cells from OD value of unstimulated control cells of the same chicken. The average SI value of 9 to 10 chickens of each group was compared among vaccine groups.
For the cell proliferation assay, PBMCs and splenocytes (1x106 cells/well in 100 µL) were suspended in E-RPMI medium and seeded in triplicate wells of 96 well flat-bottom plates (Greiner bio-one, NC). Cells were treated with OMPs (5 µg/mL in 100 µL) in E-RPMI medium and incubated for 72 h at 39°C, 5% CO2. After incubation, 20 µL of MTS+PMS solution was added and incubated for 4 h at 37°C, 5% CO2. The OD value was taken at 490 nm by the ELISA plate reader. The stimulation index (SI) was calculated by dividing OD value of stimulated cells from OD value of unstimulated control cells of the same chicken. The average SI value of 9 to 10 chickens of each group was compared among vaccine groups.
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