FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37 °C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD [32 (link)] using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
0.22 μm filter
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 0.22 μm filter is a laboratory filtration device designed to remove particles and microorganisms from liquid samples. It features a pore size of 0.22 micrometers, which is effective in trapping bacteria and other microbes while allowing the passage of smaller molecules and compounds.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 f cells, by Thermo Fisher Scientific (16 mentions)
Freestyle 293f medium, by Thermo Fisher Scientific (15 mentions)
Expifectamine 293 transfection reagent, by Thermo Fisher Scientific (15 mentions)
Nickel affinity column, by Thermo Fisher Scientific (10 mentions)
Cho k1 cells, by American Type Culture Collection (2 mentions)
Hitrap q hp 1 ml column, by GE Healthcare (2 mentions)
Psectag2 a, by Thermo Fisher Scientific (2 mentions)
Cho k1 cell, by Thermo Fisher Scientific (2 mentions)
Freestyle cho expression medium, by Thermo Fisher Scientific (2 mentions)
Hitrap, by Cytiva (2 mentions)
Harvey sterile max, by Thermo Fisher Scientific (2 mentions)
Zncl2, by Merck Group (2 mentions)
Methamphetamine, by Merck Group (1 mentions)
Varian 710 es, by Agilent Technologies (1 mentions)
Jem 1010, by JEOL (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Akta pure fast protein liquid chromatography system, by Cytiva (1 mentions)
Histrap ff 1 ml column, by Cytiva (1 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
33 protocols using 0.22 μm filter
1
SARS-CoV-2 Spike RBD Protein Production
2
Production of SARS-CoV-2 Spike RBD Protein
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FreeStyle 293 F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37 °C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDSPAGE gels and stained with Coomassie Blue.
3
Isolation of Extracellular Vesicles from Infected Macrophages
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Confluent monolayers of RAW264.7 (~ 1x107 cells) were seeded overnight in Ti175 flasks and infected with Mtb or were left un-infected. Prior to infection, the bacteria were complement opsonized in DMEM media supplemented with 10% normal horse serum for 2hrs. A MOI of 5:1 was used to obtain about ~80% of the RAW264.7 cells infected as described [10 (link), 19 (link)]. RAW264.7 cells were infected for 4 hrs, washed 3x with PBS to remove extracellular free bacteria and then cultured in DMEM media with 10% EV-free FBS (overnight spin to deplete EVs in serum) for 72hrs. EVs were isolated from the culture supernatants of infected and uninfected RAW 264.7 cells by centrifugation at 3,000×g for 10 mins to remove cell debris followed by filtration twice through 0.22μm filter (Thermo Fisher Scientific). The supernatant was further centrifuged at 100,000×g for 1hr at 4°C. The pellets were washed once and resuspended in PBS.
4
Recombinant SARS-CoV-2 RBD Protein Production
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FreeStyle 293 F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 106 cells/mL at 37 °C with 8% CO2 under regular agitation (150 rpm). Cells were transfected with the plasmid coding for SARS-CoV-2 S RBD WT using an ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant RBD proteins were purified using nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
5
Intravenous Methamphetamine Dosing Protocol
6
Synthesis and Characterization of Silica Nanoparticles
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Silica gel was prepared via a chemical neutralization reaction by mixing hydrochloric acid with a solution of reagent grade sodium silicate (SiO2 26.5%, Na2O 10.6%, and H2O 62.9%, Sigma Aldrich; Figure 1A and B ). Silica gel of 6 g synthesized from the sodium silicate solution of 23 g according to the law of conservation of mass. After thorough washing with phosphate-buffered saline and sterilization, the gel was added to 100 mL of DMEM for 48 hours. The gel-containing medium was filtered through a 0.22 μm filter (Thermo, Waltham, MA, USA). Filtered DMEM have got silica NPs (silica NP medium). Silica MPs purchased from Ditto Technology (Anyang City, Korea) were suspended same concentration of silica NP medium in DMEM (silica MP medium). The particle concentration in each type of medium was measured using an inductively coupled plasma optical emission spectrometer (Varian 710-ES, Varian, Melbourne, Australia). The size of the silica NPs was determined by transmission electron microscopy (JEM1010, JEOL Ltd, Tokyo, Japan). Basic fibroblast growth factor (Sigma-Aldrich) was used as a positive control.
7
Purification of SARS-CoV-2 Spike Receptor Binding Domain
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The CM from transfected 293F cells were collected, centrifuged at 800g for 5 minutes, filtered with a 0.22 μm filter (Thermo Fisher Scientific), and stored at –80°C before processing. Affinity purification was performed using an Akta Pure fast protein liquid chromatography system (Cytiva, formerly GE Healthcare). The S-RBD-His product (S-RBD1) purification was performed using nickel-nitrilotriacetic acid resin, where samples were supplemented to a final concentration of 30 mM imidazole (MilliporeSigma) and 1× EDTA-free protease inhibitor cocktail (Roche) before loading onto a HisTrap FF 1 mL column (Cytiva, formerly GE Healthcare), equilibrated in wash buffer (PBS 300 mM NaCl, 40 mM imidazole, pH 8.0), and eluted with a linear gradient from 40 to 500 mM imidazole. Eluted fractions were pooled, concentrated with 3000 Da (MW cutoff) ultrafiltration membrane spin column (Amicon) per the manufacturer’s instructions, dialyzed into PBS, and stored at –80°C. The purified S-RBD1 was quantified using NanoDrop OneC (280 nm absorbance, Thermo Fisher Scientific), BCA assay (Thermo Fisher Scientific) relative to bovine serum albumin (BSA) standard, and/or Qubit 3 Fluorometer (Qubit Protein Assay, Thermo Fisher Scientific) according to the manufacturer’s instructions. The reagents for the protein purifications are listed in Supplemental Table 1 .
8
Recombinant SARS-CoV-2 RBD Protein Production
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FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37°C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S WT RBD (Beaudoin-Bussières et al., 2020 (link)) using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
9
Gel Filtration Chromatography of Honey Proteins
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The
extracted crude honey proteins were subjected to gel filtration chromatography
(GFC) on fast protein liquid chromatography (FPLC) utilizing an AKTA
pure system (GE Healthcare, Uppsala, Sweden). The gel filtration column
Hi-prep 26/60 Sephacryl S-200 column (GE-Healthcare, Uppsala, Sweden)
was used. The system was equilibrated with buffer A as the mobile
phase. The extracted crude honey proteins dissolved in buffer A were
filtered by a 0.22 μm filter (Thermo Scientific, USA) and were
applied on to the column with an eluted flow rate of 1 mL/min. The
fractions were collected as 2 mL/tube.
extracted crude honey proteins were subjected to gel filtration chromatography
(GFC) on fast protein liquid chromatography (FPLC) utilizing an AKTA
pure system (GE Healthcare, Uppsala, Sweden). The gel filtration column
Hi-prep 26/60 Sephacryl S-200 column (GE-Healthcare, Uppsala, Sweden)
was used. The system was equilibrated with buffer A as the mobile
phase. The extracted crude honey proteins dissolved in buffer A were
filtered by a 0.22 μm filter (Thermo Scientific, USA) and were
applied on to the column with an eluted flow rate of 1 mL/min. The
fractions were collected as 2 mL/tube.
10
Producing Recombinant SARS-CoV-2 RBD Protein
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FreeStyle 293F cells (Thermo Fisher Scientific) were grown in FreeStyle 293F medium (Thermo Fisher Scientific) to a density of 1 × 106 cells/mL at 37°C with 8 % CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen) (Beaudoin-Bussieres et al., 2020 ; Prevost et al., 2020 ). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Thermo Fisher Scientific). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
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