Before testing, the cell culture broth pH was measured. All experiments were performed in 96‐deep‐well plates. Dilutions from 1 to 12 mM of ZnCl2 were prepared from a 100 mM solution and then added to 0.5 mL of the cell culture broth containing the antibody. After 20 min of incubation at room temperature on the end‐over‐end shaker (Stuart rotator SB3; Cole‐ Parmer, Vernon Hills, IL), the plate was centrifuged at 4000 rcf for 10 min (Centrifuge Heraeus Multifuge X3, Rotor HIGHPlateTM 6000; Thermo Fisher Scientific, Waltham, MA). The supernatant was withdrawn and filtered through 0.2 µm filters (0.2 µm GHP AcroPrepTM 96 filter plate; Pall Life Sciences, Ann Arbor, MI) and analyzed with protein A affinity chromatography (described in Section 2.3).
Stuart rotator sb3
Manufactured by Cole-Parmer
Sourced in United Kingdom
The Stuart rotator SB3 is a compact and versatile laboratory equipment designed for gentle mixing and rotation of samples. It features a variable rotational speed range and can accommodate a variety of sample containers, making it suitable for a wide range of laboratory applications.
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Lab products found in correlation
6 protocols using stuart rotator sb3
1
Antibody Purification via Zinc Chloride
2
Trastuzumab Capture by PEG6000
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Increasing concentrations of PEG6000 in the range of 0 to 15% (w/w) were added to CCCS from a 50% stock solution of PEG6000 in 100 mM MOPS pH 7.0. CCCS was alternatively pre‐treated with a 100 mM of CaCl2 final concentration to assess the impact of CaCl2 precipitation on trastuzumab capture by PEG6000. Experiments were performed in 1.5 mL centrifuge tubes with 1 mL working volume. After addition of PEG6000, samples were incubated for 15 min on the end‐over‐end shaker (Stuart rotator SB3; Cole‐ Parmer, Vernon Hills, IL). For subsequent analysis, samples were centrifuged at 2000 g for 2 min and the supernatant was collected in fresh centrifuge tubes.
3
Optimizing hcDNA Precipitation with CaCl2
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To define the best conditions for efficient hcDNA precipitation, increasing concentrations of CaCl2 from 0 to 350 mM were added to CCCS from a 1 M stock solution of CaCl2 in 100 mM MOPS, pH 7.0. The screening was performed in 1.5 mL centrifuge tubes with a total working volume of 1 mL. Samples were gently agitated for 5 min on the end‐over‐end shaker (Stuart rotator SB3; Cole‐ Parmer, Vernon Hills, IL). This time was sufficient for the maximal hcDNA precipitation at the corresponding CaCl2 concentration. After precipitation, samples were centrifuged at 6000 g for 6 min and the supernatant was collected in fresh centrifuge tubes for subsequent analysis.
4
Trastuzumab Redissolution and Neutralization
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Redissolution of captured trastuzumab was performed batch‐wise with a 50 mM phosphate buffer pH 2.5 at a volumetric dilution sample:buffer of 1:5. Samples were incubated for 60 min at low pH (<3.5) on the end‐over‐end shaker (Stuart rotator SB3; Cole‐ Parmer, Vernon Hills, IL) and neutralized by addition of 200 mM phosphate buffer pH 8.0 with a volumetric dilution sample:buffer of 4:1. This ratio resulted in a solution with final pH 6.5. Samples were then immediately analyzed, stored at 4°C or frozen at −20°C for prolonged storage.
5
Trimethoprim Release from Microparticles
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Release studies were performed for
2 weeks at 4 °C, room temperature,
and 37 °C either in artificial urine or HEPES/PLU as a dispersion
medium. Each sample was a dispersion of 2.0–3.0 mg lyophilized
microparticles in 1.5 mL of medium. For analysis at 4 °C, the
samples were stirred at 500 rpm in a fridge. Other samples were placed
in a tube rotator (Stuart Rotator SB3, Cole-Parmer, Staffordshire,
U.K.) either at room temperature or at 37 °C. Trimethoprim release
was analyzed daily: the particle suspension was centrifuged (14,000
rpm, 10 min, 4 °C) and 1 mL of the supernatant was withdrawn,
lyophilized, and quantified by HPLC, as described above. Samples were
immediately replaced by adding 1 mL of fresh medium. After 2 weeks,
the remaining microparticles were dissolved in 2 mL of ethyl acetate
(seeSection 4.3.2.1 ), and their trimethoprim content was determined as described
above.
Drug release data were fitted to the pharmacokinetic
models depicted below:36 (link),37 (link)zero order first order Higuchi Korsmeyer–Peppas Weibull model
The release curves were correlated to the models
and evaluated
by calculating the adjusted coefficient of determination (R2-adjusted)37 (link) where n is the number of
dissolution data points (M/t), and p is the number of parameters in the model.
2 weeks at 4 °C, room temperature,
and 37 °C either in artificial urine or HEPES/PLU as a dispersion
medium. Each sample was a dispersion of 2.0–3.0 mg lyophilized
microparticles in 1.5 mL of medium. For analysis at 4 °C, the
samples were stirred at 500 rpm in a fridge. Other samples were placed
in a tube rotator (Stuart Rotator SB3, Cole-Parmer, Staffordshire,
U.K.) either at room temperature or at 37 °C. Trimethoprim release
was analyzed daily: the particle suspension was centrifuged (14,000
rpm, 10 min, 4 °C) and 1 mL of the supernatant was withdrawn,
lyophilized, and quantified by HPLC, as described above. Samples were
immediately replaced by adding 1 mL of fresh medium. After 2 weeks,
the remaining microparticles were dissolved in 2 mL of ethyl acetate
(see
above.
Drug release data were fitted to the pharmacokinetic
models depicted below:36 (link),37 (link)zero order first order Higuchi Korsmeyer–Peppas Weibull model
The release curves were correlated to the models
and evaluated
by calculating the adjusted coefficient of determination (R2-adjusted)37 (link) where n is the number of
dissolution data points (M/t), and p is the number of parameters in the model.
6
Microplastics Exposure Impacts on Oyster Gametes
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Oyster' gametes from males (n=6) and females (n=6) were individually exposed to NPs.
Four different concentrations of PS-COOH or PS-NH 2 were tested separately on oocytes and spermatozoa: Control (no NPs), 0.1 , 1, 10 and 100 mg L -1 of PS-COOH or PS-NH 2 which correspond to 0, 1.9×10 1 , 1.9×10 2 , 1.9×10 3 and 1.9×10 4 particles spermatozoa -1 ; and 1.9×10 3 , 1.9×10 4 , 1.9×10 5 and 1.9×10 6 particles oocyte -1 , respectively. Samples were kept in continuous movement (program 3) using a Stuart rotator SB3 (Cole-Parmer, UK) in order to prevent cell sedimentation, in a dark room at 18ºC. Sampling was performed in fresh samples after 1, 3 and 5 h exposure to NPs. Additionally, after
3h exposure, control and exposed samples of oocytes and spermatozoa were fixed in formaldehyde (3% final) for later microscopy observations. 2.4. Analyses 2.4.1. Microscopy
Four different concentrations of PS-COOH or PS-NH 2 were tested separately on oocytes and spermatozoa: Control (no NPs), 0.1 , 1, 10 and 100 mg L -1 of PS-COOH or PS-NH 2 which correspond to 0, 1.9×10 1 , 1.9×10 2 , 1.9×10 3 and 1.9×10 4 particles spermatozoa -1 ; and 1.9×10 3 , 1.9×10 4 , 1.9×10 5 and 1.9×10 6 particles oocyte -1 , respectively. Samples were kept in continuous movement (program 3) using a Stuart rotator SB3 (Cole-Parmer, UK) in order to prevent cell sedimentation, in a dark room at 18ºC. Sampling was performed in fresh samples after 1, 3 and 5 h exposure to NPs. Additionally, after
3h exposure, control and exposed samples of oocytes and spermatozoa were fixed in formaldehyde (3% final) for later microscopy observations. 2.4. Analyses 2.4.1. Microscopy
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