Prior to UPLC/MS analysis, serum samples were thawed at room temperature and an aliquot of 100 ml of serum sample was mixed with 300 ml methanol. The mixture was vibrated for 30s and then left to stand for 45 min at room temperature. The mixture was then centrifuged at 4°C at 10,000 × g for 30 min. The supernatant was filtered using a 0.22 µm membrane (Merck Millipore, Darmstadt, Germany).
0.22 m membrane
Manufactured by Merck Group
Sourced in United States, Germany, Brazil
The 0.22 µm membrane is a laboratory filtration device designed for the removal of microorganisms from liquids. It features a pore size of 0.22 micrometers, which is effective in retaining bacteria and other particulates while allowing the passage of dissolved substances.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Q700 sonicator, by Qsonica (1 mentions)
Histrap hp, by Cytiva (1 mentions)
Ir prestige 21, by Shimadzu (1 mentions)
Bicinchoninic acid protein assay reagent, by Beyotime (1 mentions)
Nitrocellulose membrane, by Beyotime (1 mentions)
Tween 20, by Merck Group (1 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Denosumab, by Amgen (1 mentions)
Igepal ca 630 np 40, by Merck Group (1 mentions)
Sep pak light c18 cartridges, by Waters Corporation (1 mentions)
Nisin, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Spectramax gemini xps fluorescence microplate reader, by Molecular Devices (1 mentions)
Uv 2102pc, by Unico (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Cd cho, by Thermo Fisher Scientific (1 mentions)
48 protocols using 0.22 m membrane
1
Serum metabolite extraction for UPLC/MS
2
Purification of Recombinant MSP2N2 Protein
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The method for purification of MSP2N2 was adapted from a published protocol34 . Cell suspensions were thawed and lysed by sonication on ice using a Q700 sonicator (Qsonica) (50% output amplitude, 30 cycles of 10 s on, 20 s off). The cell lysate was clarified by centrifugation using a SS34 rotor at 30,000 × g for 1 h at 4 °C. The supernatant was collected, syringe filtered through a 0.22 µm membrane (Merck Millipore Ltd.) and applied to a 1 mL Ni-NTA column (His-TrapTM HP, Cytiva) equilibrated with 50 mM Tris-HCl (pH 8), 500 mM NaCl (=buffer A) + 1% v/v Triton X-100. The column was washed with 10 column volumes of buffer A + 1% v/v Triton X-100 followed by 10 column volumes of buffer A + 50 mM sodium cholate. Non-specifically bound proteins were washed with 10 column volumes of buffer A + 80 mM imidazole and finally MSP2N2 was eluted with buffer A + 400 mM imidazole. To obtain highly pure MSP2N2, the eluate from 1 mL Ni-NTA column was reapplied to a 5 mL Ni-NTA column (His-TrapTM HP, Cytiva) and the same procedure was repeated. Pure MSP2N2 fractions were pooled and dialysed against 2 L of 10 mM MOPS (pH 7.5 at 4 °C), 50 mM KCl at 4 °C. Sample homogeneity was confirmed by SDS-PAGE, and the protein flash frozen and stored at −80 °C.
3
Herbal Decoction Formulation and Preparation
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The BFD decoction is composed of 6 medicinal herbs: milkvetch root (Huang-Qi), Radix asteris (Zi-Yuan), Cortex Mori Radicis (Sang-Bai-Pi), Rehmannia glutinosa (Di-Huang), Codonopsis pilosula (Dang-shen) and Schisandra chinensis (Wu-Wei-Zi), Hedyotic diffusa (Bai-Hua-She-She-Cao), Duchesnea (She-Mei) and Scutellaria barbata (Ban-Zhi-Lian) were added to make MBFD. The components of BFD and MBFD were converted into formula granules by Beijing Tcmages Pharmaceuticals Co., Ltd. (Beijing, China) at a receiving rate of 18.8 and 14.1%, respectively. The quality of the BFD and MBFD granules was monitored by Fourier transform infrared spectroscopy (model IRPRestige-21; Shimadzu Corp., Kyoto, Japan). Prior to use, the formula granules were dissolved in deionized water at about 50°C, centrifuged at 13,800 × g for 30 min to remove drug sediment, and sterilized by filtration through a 0.22 µm membrane (EMD Millipore, Billerica, MA, USA); finally, the solutions were stored at −80°C. The concentrations of BFD and MBFD indicated in the subsequent text denote crude drug concentrations.
4
Extraction and Characterization of Elderberry Powder
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The fruits of elderberry (Sambucus nigra L.) cultivar Sampo, obtained from Bio Berry Poland (Warsaw, Poland), were homogenized to fruit pulp, which was subsequently frozen at −80 °C and subjected to freeze-drying at a vacuum pressure of 0.1 mbar and temperature of 20 °C for 23 h and post-drying at 23 °C for 3 h using a freeze dryer (LMC-1, Martin Christ Gefriertrocknungsanlagen GmbH, Germany). The lyophilized EDB were finely ground and packaged under nitrogen atmosphere. The EDB extract was obtained by dissolving the EDB powder in complete culture medium with the pH adjustment to 7.4. The EDB suspension was then centrifuged (3000 g, 5 min) and filtered through a 0.22 µm membrane (Merck, Germany).
5
Western Blot Analysis of PTEN in MSCs and CM
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Briefly, the MSCs were washed with PBS three times and collected using cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Cell lysates were incubated on ice for 30 min. The protein concentration was determined using bicinchoninic acid protein assay reagents (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Equal amounts of protein (50 µg/sample) were loaded on each lane and separated by electrophoresis in 12% sodium dodecyl sulfate polyacrylamide gel and electrotransferred to nitrocellulose membranes (Beyotime Institute of Biotechnology). The membrane was incubated in blocking buffer for 1 h at RT and then incubated overnight at 4°C with monoclonal rabbit anti-mouse PTEN primary antibodies (cat. no. 217702; dilution, 1:1,000; R&D Systems, Inc., Minneapolis, MN, USA). The blots were rinsed with Tris-buffered saline and Tween-20 (EMD Millipore, Billerica, MA, USA) three times, incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (cat. no. A0216; dilution, 1:1,000; Beyotime Institute of Biotechnology) for 60 min and detected by chemiluminescence using ECL Hyperfilm (EMD Millipore). The CM samples were filtered through a 0.22 µm membrane (EMD Millipore) and equally concentrated using a 10,000 molecular weight cut-off (catalog no., 42406; EMD Millipore). Then, PTEN was detected in the CM.
6
Production and Purification of Therapeutic Antibodies
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IgG1, Adalimumab (pI 8.2) was produced in Chinese hamster ovary (CHO) cells in fed‐batch fermentation. Trastuzumab (pI 8.4) was also produced in CHO cells but a perfusion system. IgG2 Denosumab (pI 8.3) was bought from Amgen (California, USA). For primary clarification, cells were removed by centrifugation and the host cell broth was filtered with a 0.22 µm membrane (Merck KGaA, Darmstadt, Germany), having, in the end, a host cell clarified broth (HCCB).
7
HEV Particle Purification and Characterization
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Supernatants of PLC/PRF/5 cells infected with HEV-A4 were combined into an 80 mL stock. The stock was ultracentrifuged for 16 h at 160,000× g. The precipitate was re-suspended in 50 mM Tris-HCl buffer (pH 7.6; TB) (Sigma-Aldrich) and split into 1 mL aliquots. The samples were subjected to one of the following treatments: (i) 4 cycles of freezing and thawing; (ii) incubation with 2% IGEPAL® CA-630 (NP-40) (Sigma-Aldrich) for 2 h at 37 °C, as per Qi et al.; (iii) sonication at 40 kHz for 4 min in ice water filtered through a 0.22 µm membrane (Merck, New Jersey, USA), and suspended in TB with 10% sodium deoxycholate (NaDOC/T) (Merck) and 1% EDTA trypsin for 2 h at 37 °C, as per Ideno et al.; or (iv) no treatment [24 (link),25 (link)]. These are summarized in Table 2 .
HEV-A patient stool filtrate and serum were used as controls, representing nHEV and eHEV, respectively. We loaded 1 mL of each sample into an ultracentrifugation tube, and the samples passed through an iodixanol gradient (2 mL each of 60%, 50%, 40%, 30%, 20%, and 10%) (Abbot, Chicago, IL, USA). The tubes were centrifuged at 160,000× g at 4 °C for 16 h. Twelve fractions of 1 mL were recovered, and the density and HEV RNA were measured for each fraction via refractometry and qRT-PCR, respectively.
HEV-A patient stool filtrate and serum were used as controls, representing nHEV and eHEV, respectively. We loaded 1 mL of each sample into an ultracentrifugation tube, and the samples passed through an iodixanol gradient (2 mL each of 60%, 50%, 40%, 30%, 20%, and 10%) (Abbot, Chicago, IL, USA). The tubes were centrifuged at 160,000× g at 4 °C for 16 h. Twelve fractions of 1 mL were recovered, and the density and HEV RNA were measured for each fraction via refractometry and qRT-PCR, respectively.
8
Investigating Fungal Spore Morphology Modulation
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The methodology used for observing the influence of the cell-free supernatant of Z. sp. ISTPL4 on the spores of S. indica was taken from Bandyopadhyay et al. (2016) . For this, a bacterial culture of Z. sp. ISTPL4 was grown in a LB medium and added to HK minimal medium for 96 hours at 28°C–30°C, with shaking at 120 rpm, followed by centrifugation of bacterial culture at 10,000 rpm to obtain a cell-free supernatant (Labogene, Denmark). The resulting cell-free supernatant was filtered via a 0.22-µm membrane (Millipore) and then inoculated in fungal spores (4.85 × 105 spores/mL) for an incubation period of 12 hours. The spore morphology of S. indica was observed under a Nikon confocal microscope (model Nikon A1) at 60× magnification.
9
Radiolabeling of DOTA-iFAP with Ga-68
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68Ga-iFAP was prepared using a synthesis module designed to label biomolecules with 68Ga (iQS® Ga-68 Fluidic Labeling Module; ITM; Munich, Bavaria, Germany) for clinical use (GMP conditions). Briefly, 25 µg of DOTA-iFAP dissolved in 1 mL of 0.25 M sodium acetate, pH 4.5, was added to the system reactor and heated for 5 min (105–110 °C). Then, 5 mL of 68GaCl3 in 0.05 M HCl freshly eluted from the generator (68Ge/68Ga generator; ITM; Munich, Germany) was added and heated for 12 min (105–110 °C). Finally, it was purified using C18 Sep-Pak Light cartridges (solid phase extraction; Waters; Milford, MA, USA) and sterilized by a 0.22 µm membrane (Millipore; Burlington, MA, USA). The 68Ga-iFAP solution was assayed for radiochemical purity, sterility, and bacterial endotoxins following the general methods of the Mexican Pharmacopoeia [11 ].
10
Standard Nisin Quantification Protocol
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The standard nisin (Sigma-Aldrich, St. Louis, MO, USA—containing 2.5% nisin, with 1,000,000 IU/g in its composition) solution was prepared by dissolving 1 g of nisin in 10 mL of phosphate buffer solution (PBS-pH 7.0), as in Table 1 . The solution was centrifuged at 13,201 g for 10 min at 10 °C and the supernatant was collected and filtered in a 0.22 µm membrane (Millipore, Burlington, MA, USA). The nisin standard curve was evaluated by an agar diffusion assay. The nisin bioindicator Lactobacillus sakei ATCC 15521 was used for the agar diffusion assay [6 (link),23 (link)].
The concentrations of standard nisin were related by the diameter of the inhibition halo (H, mm), and the activity of nisin was determined and expressed in arbitrary units per mL (AU/mL). The activity of nisin was based on the dilution of the standard nisin calibration curves. The correlation between AU/mL and international units per mL (IU/mL) was 1.09 ± 0.17 AU to 1.0 IU (40 IU = 1 µg of pure nisin A) [14 (link),26 (link)].
The concentrations of standard nisin were related by the diameter of the inhibition halo (H, mm), and the activity of nisin was determined and expressed in arbitrary units per mL (AU/mL). The activity of nisin was based on the dilution of the standard nisin calibration curves. The correlation between AU/mL and international units per mL (IU/mL) was 1.09 ± 0.17 AU to 1.0 IU (40 IU = 1 µg of pure nisin A) [14 (link),26 (link)].
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