PLGA NP with entrapped OVA alone or in combination with R848, poly I:C or α-GalCer were prepared using an o/w emulsion and solvent evaporation–extraction method as previously described.33 (link) In brief, 100 mg of PLGA in 3 mL of dichloromethane containing, 5 mg OVA, 1.6mg SIINFEKL or HPV16 E7, 2 mg poly I:C, 0.5 mg R848 or 0.1 mg α-GalCer and 0.5mg Atto647N was added dropwise to 25mL of aqueous 2% polyvinyl alcohol and emulsified for 120 seconds using a digital sonicator from Branson Ultrasonics (Danbury, CT). The solvent was evaporated overnight at 4°C and NP were collected by centrifugation at 14.000 rpm for 20 min, washed six times with distilled water and lyophilized.
Digital sonicator
Manufactured by Emerson
Sourced in United States
The Digital Sonicator is a laboratory equipment designed for the controlled application of ultrasonic energy. It generates high-frequency sound waves to disrupt and homogenize samples, facilitate extraction processes, and aid in various laboratory techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
T bet, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Foxo1, by Santa Cruz Biotechnology (2 mentions)
Nupage precast gels, by Bio-Rad (2 mentions)
Mini protean tetra cell system, by Cytiva (2 mentions)
4ebp1 p s37 s46, by Cell Signaling Technology (2 mentions)
4ebp1 ps65, by Fortis Life Sciences (2 mentions)
S6k p t389, by Fortis Life Sciences (2 mentions)
Akt p t308, by Fortis Life Sciences (2 mentions)
Akt p s473, by Fortis Life Sciences (2 mentions)
Foxo1 3a p t24 32, by Santa Cruz Biotechnology (2 mentions)
X ray film, by Konica Minolta (2 mentions)
Thermomixer, by Thermo Fisher Scientific (1 mentions)
Triglyceride quantification kit, by Abcam (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Complete mini, by Roche (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Savant spd121p, by Thermo Fisher Scientific (1 mentions)
Stereomicroscope, by Zeiss (1 mentions)
7 protocols using digital sonicator
1
PLGA Nanoparticles for Vaccine Delivery
2
Quantifying Nematode Triglycerides with CTE
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Total TGs were quantified in age-synchronized worm nematodes cultured in NGM plates seeded with E. coli OP50 using the Triglyceride Quantification Kit (Biovision; Mountain View, CA, USA). For the treatments, NGM plates were supplemented with 2 mg/mL of CTE or Orlistat (6 µg/mL) as a positive control. Worms at young adult stage were then collected and washed with M9 buffer. Supernatant was removed after worm setting, and 400 µL of triglyceride assay buffer was added to worm pellet. Worms were sonicated with a digital sonicator (Branson Ultrasonics Corp.; Danbury, CT, USA) at 10% of power for 30 s. To solubilize all TGs in the solution, samples were slowly heated twice at 90 °C for 5 min in a thermomixer (Thermo Fisher; Waltham, MA, USA). After centrifugation, 50 µL aliquots were used for the triglyceride assay following the manufacturer’s instructions. Protein content was measured using BCA Protein Assay Kit (Thermo Scientific; Rockford, IL, USA). Four different biological replicates were included for each condition in two independent experiments.
3
Western Blot Analysis of Signaling Proteins
4
Cell Lysis and DNA Fragmentation Protocol
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Frozen cell pellets were resuspended in 1x IPOD lysis buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl) containing 1x protease inhibitors (Roche Complete Mini, EDTA free, Roche Diagnostics GmbH, Mannheim, Germany) and 52.5 kU/mL of ready-lyse (Epicentre, Madison, WI): 600 μL per pellet (stationary phase cells were diluted 10× prior to lysis, and only 1/10 of the resulting material used, due to the much higher biomass of those pellets). We incubated the resuspended pellet for 15 minutes at 30°C and then placed it on ice. We then sonicated the cells using a Branson digital sonicator at 25% power, using three 10-second bursts with 10-second pauses between bursts. The cells were maintained in a wet ice bath throughout sonication.
We then performed a calibrated DNA digestion to sub-200-bp fragments, by adding to the sonicated lysates 60 μg RNase A (Thermo Fisher Scientific, Waltham, MA), 6 μL DNase I (Fisher product #89835), 5.4 μL 100 mM MnCl2, and 4.5 μL 100 mM CaCl2, and then incubating on ice. While the appropriate digestion time must be calibrated for each particular sample type and batch of DNase, 30 minutes of digestion proved appropriate for all samples here. Reactions were quenched after completion by the addition of 50 μL 500 mM EDTA (pH 8.0), typically yielding 50- to 200-bp fragments.
We then performed a calibrated DNA digestion to sub-200-bp fragments, by adding to the sonicated lysates 60 μg RNase A (Thermo Fisher Scientific, Waltham, MA), 6 μL DNase I (Fisher product #89835), 5.4 μL 100 mM MnCl2, and 4.5 μL 100 mM CaCl2, and then incubating on ice. While the appropriate digestion time must be calibrated for each particular sample type and batch of DNase, 30 minutes of digestion proved appropriate for all samples here. Reactions were quenched after completion by the addition of 50 μL 500 mM EDTA (pH 8.0), typically yielding 50- to 200-bp fragments.
5
Western Blot Analysis of Signaling Proteins
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Cells (20 × 106) were lysed in RIPA buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM TCEP (Pierce), protease and phosphatase inhibitors (Roche). Lysates were sonicated in a Branson Digital sonicator on ice, centrifuged (4 °C, 16,000 × g for 10 min). Samples were adjusted to 1× LDS sample buffer (life technologies) and 25 mM TCEP was added prior boiling for 10 min. Each lane was loaded with the equivalent of 140,000 CTLs and separated by SDS-PAGE (life technologies NuPAGE precast gels or Bio-Rad Mini-PROTEAN tetra cell system) and transferred to nitrocellulose membranes (Whatman). Blots were probed with the following antibodies: 4EBP1 p-S37/S46 (Cell Signaling Technology (CST) #2855), 4EBP1 - pS65 (CST #9451), 4EBP1 (CST #9452), S6K p-T389 (CST #9239), S6K (CST #9202), Akt p-T308 (CST #4056), Akt p-S473 (CST #4058), SMC1 (Bethyl Laboratories, A300-055A), T-bet (eBioscience 14-5825), IRS2 (CST #4502), PTEN (Santa Cruz sc-7974), FOXO1/3A p-T24/32 (CST #9464), FOXO1 (CST #9454). X-ray films (Konica) were used to monitor chemiluminescence reactions catalysed by HRP-conjugated secondary antibodies. All immunoblots shown are representative of 3 or more biological replicates.
6
Preparation of PLGA Nanoparticles by Double Emulsion
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PLGA nanoparticles were prepared by double emulsion based on a previously described procedure [18 (link)]. Briefly, PLGA (PLGA20: 20 mg; PLGA60: 60 mg) was dissolved in 2 mL of ethyl acetate and sonicated for 15 s (70% amplitude) using an ultrasonic homogenizer (Branson Digital Sonicator, Saint Louis, MO, USA), resulting in a w/o emulsion. An equal volume of PVA solution (7% (w/v) in water) was added and sonicated for an additional 30 s (70% amplitude), resulting in a w/o/w double emulsion. The organic solvent was evaporated in a Savant SPD121P vacuum centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 10,000 rpm for approximately 1 h at 40 °C. Fluorescent nanoparticles were prepared, adding rhodamine (0.5 mg·mL−1, 1 mg) to the organic solution. To prepare peptide-loaded nanoparticles, 400 µg of PLP139-151 was added to the PLGA organic solution. The prepared nanoparticle suspensions were stored at 4 °C until further use.
7
Biofilm Formation and Quantification
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Cells from a single colony isolated on LB plates were grown to mid-logarithmic phase in 2-ml of LB (~3 hours at 37°C). Cells were diluted 1:1000 and were added to the 2 mL liquid MSgg in 24 well plates (Thermo Scientific). The cultures were then grown at 30°C for 72 h in the dark. The pellicles were imaged at 72 h. Cells were either grown in the presence or absence of CM as indicated in each corresponding figure legends. The pellicles were imaged using stereomicroscope (Zeiss), using Objective Plain 0.5× FWD 134 mm lens at 10x magnification.
Captured images were processed using Zen software (Zeiss).
For CFU count, pellicles were harvested at 72 h by scrapping off the entire pellicle plus pipetting the entire underlying cell suspension, and suspending in PBS (phosphate-buffered saline). This was followed by sonication using a BRANSON digital sonicator, at an amplitude of 10% and a pulse of 5 sec to separate the cells without compromising their viability. 200 µl of sonicated cells suspension was transferred to a Griener 90 well plate (Sigma-Aldrich) and a serial dilution ranging from 10 -1 to 10 -7 was performed in PBS. From the dilutions 20 µl of sample was transferred on a LB plate, incubated at 30 °C overnight and then counted.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Captured images were processed using Zen software (Zeiss).
For CFU count, pellicles were harvested at 72 h by scrapping off the entire pellicle plus pipetting the entire underlying cell suspension, and suspending in PBS (phosphate-buffered saline). This was followed by sonication using a BRANSON digital sonicator, at an amplitude of 10% and a pulse of 5 sec to separate the cells without compromising their viability. 200 µl of sonicated cells suspension was transferred to a Griener 90 well plate (Sigma-Aldrich) and a serial dilution ranging from 10 -1 to 10 -7 was performed in PBS. From the dilutions 20 µl of sample was transferred on a LB plate, incubated at 30 °C overnight and then counted.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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