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Plate thermoshaker

Manufactured by Biosan
Sourced in Latvia

The Plate Thermoshaker is a laboratory instrument designed to provide simultaneous heating and shaking functions for microplates or deep-well plates. The device maintains a consistent temperature across the entire plate while gently agitating the samples, facilitating efficient incubation and mixing processes.

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7 protocols using plate thermoshaker

1

FVIII Aggregation Kinetics Evaluation

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To prepare seeds, FVIII samples (0.122 μM) were incubated for 2, 5, 8 or 18 h at 45°C in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker (Biosan). Native FVIII samples (0.122 μM) were mixed 1:1 with the corresponding seeds and time-dependent aggregation at 45°C was initiated. Samples were stored at −80°C until HPLC-SEC analysis.
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2

Peptide Antimicrobial Potency Screening

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Bacterial test cultures were grown in the Mueller-Hinton broth (MHB, Sigma, St. Louis, MO, USA) at 37 °C to mid-log phase and then diluted with the 2× MHB media supplemented with 1.8% NaCl or without salt to reach a final cell concentration of 106 CFU/mL. An amount of 50 µL of the obtained bacterial suspension was added to aliquots of 50 µL of the peptide solutions serially diluted with sterilized 0.1% bovine serum albumin (BSA) in 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011, Hamburg, Germany). After incubation for 24 h at 37 °C and 950 rpm on the plate thermoshaker (Biosan, Riga, Latvia), minimum inhibitory concentrations (MIC) were determined as the lowest peptide concentrations that prevented growth of a test microorganism observed as visible turbidity. In most cases, no significant divergence of MIC values was observed (within ±1 dilution step). The results were expressed as the median values determined on the basis of at least three independent experiments performed in triplicate.
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3

Antimicrobial Peptide Screening Protocol

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The antimicrobial activity of investigated peptides was determined by the method of two-fold serial dilutions in the sterile 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011, Hamburg, Germany). Mueller–Hinton broth was used as a culture fluid. The bacterial strains were cultured in the MH medium at 37 °C for 16 h, then aliquots of culture were diluted with fresh MH and grown to optical density (OD600) of 1. The bacterial cultures were prepared in the NaCl-enriched MH broth (42 g/L Mueller–Hinton broth, 18 g/L NaCl) to the final concentration of 1 × 106 colony-forming units (CFU)/mL. In the next step, 50 μL of obtained cell culture was added to 50 μL of peptides to reach the final cell density of 5 × 105 CFU/mL; the compounds had been previously diluted with sterilized 0.1% BSA. After adding the suspension of cells, the plate was incubated at 37 °C for 24 h and 1000 rpm on the plate thermo-shaker ((Biosan, Riga, Latvia). The values of MIC (minimum inhibitory concentration) were identified as the lowest concentration of peptide at which the growth of the culture had not taken place and defined visually, or 0.1 mg/mL of resazurin was added to each well and incubated at 37 °C for 2 h. The tests were carried out at no fewer than two repetitions of free independent experiments, and the MIC of peptides was calculated as an averaged value of these assays.
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4

FVIII Thermal Stability Assay

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All FVIII samples (0.122 μM) were incubated at 45°C for 24 h in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker (Biosan, Riga, Latvia). Samples were withdrawn after various time intervals and immediately frozen at −80°C until HPLC-SEC analysis.
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5

Antimicrobial Activities Assessment of Recombinant Peptides

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Bacterial test cultures were grown in the Mueller-Hinton (MH) medium at 37 °C to mid-log phase and then diluted with the 2 x MH medium supplemented with 1.8% NaCl. 50 µL of the bacterial suspension with a final cell concentration of 106 CFU/mL were added to aliquots of 50 µL of the peptide solutions and serially diluted with sterilized 0.1% bovine serum albumin in 96-well flat-bottom polystyrene microplates (Eppendorf, Hamburg, Germany). Bacterial cells were incubated for 24 h at 37 °C and 900 rpm on the plate thermoshaker (Biosan, Riga, Latvia). The minimum inhibitory concentrations (MIC) were determined as the lowest peptide concentration that prevented growth of a test microorganism observed as visible turbidity. The results were expressed as median values of three independent triplicated experiments. Assessment of antimicrobial activities of the recombinant PM III, other polyphemusins, and tachyplesins was conducted against Gram-negative (Escherichia coli ML-35p, Klebsiella pneumoniae (clinical isolate, CI 287), Pseudomonas aeruginosa PAO1) and Gram-positive (Staphylococcus aureus ATCC 29213, Staphylococcus aureus 209P, Bacillus subtilis B-886, Micrococcus luteus B-1314) bacteria.
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6

Antimicrobial Activity Assessment of Peptides

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The antimicrobial activity of the peptides was determined via the method of two-fold serial dilutions in sterile 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011) in Mueller–Hinton broth (MHB, Sigma, Kawasaki, Kanagawa, Japan) supplemented with 0.9% NaCl [32 (link)]. Bacterial cultures were grown in MHB at 37 °C until the culture reached an optical density OD600 of 1.0 and diluted with the broth so that to reach a final cell concentration of 106 CFU/mL. Then 50 μL of the test culture were added to 50 μL of aqueous solutions of the peptides, previously diluted in 0.1% sterilized bovine serum albumin (BSA) to reduce non-specific binding of the peptides to the surface of the plates. Next, the plate was incubated for 24 h at 37 °C and 950 rpm on the plate thermoshaker (Biosan). The values of the minimum inhibitory concentrations (MIC) were determined as the minimum concentration of the peptide, at which there is no culture growth. The results were expressed as the median values of three independent experiments performed in triplicate.
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7

Antimicrobial Peptide Activity Assay

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Bacterial test cultures were grown in the Mueller-Hinton broth (MH; Sigma, St. Louis, MO, USA) at 37 °C to mid-log phase and then diluted with the 2× MH medium supplemented with 1.8% NaCl or without salt so that to reach a final cell concentration of 106 CFU/mL. 50 µL of the obtained bacterial suspension were added to aliquots of 50 µL of the peptide solutions serially diluted with sterilized 0.1% bovine serum albumin (BSA) in 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011, Hamburg, Germany). After incubation for 24 h at 37 °C and 950 rpm on the plate thermoshaker (Biosan, Riga, Latvia), minimum inhibitory concentrations (MIC) were determined as the lowest peptide concentrations that prevented the growth of a test microorganism observed as visible turbidity, and the absorbance at 620 nm. To verify MIC values, the respiratory activity of the bacteria was determined. Briefly, 20 µL of 0.1 mg/mL resazurin (a redox indicator; Sigma, St. Louis, MO, USA) was added to the wells after 24 h of incubation, and the plate was incubated for an additional two hours. The reduction of resazurin to resorufin was measured. In most cases, no significant divergence of MIC values was observed (within ±1 dilution step). The results were expressed as the median values determined based on at least three independent experiments performed in triplicate.
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