MSC CM was concentrated from 500 μL to 50 μL by Amicon Ultra filters (UFC5003, MilliporeSigma). The concentrated CM with IgG or anti-Cyr61 antibody was mixed with Matrigel (356231, Corning) to a final volume of 500 μL on ice, and the mixture was then implanted subcutaneously in the dorsal area of Plg−/− mice. Seven days after implantation, the Matrigel plugs were collected and embedded in paraffin for immunostaining.
Amicon ultra filter
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy
Amicon Ultra filters are laboratory centrifugal devices used for concentrating and purifying macromolecules such as proteins, peptides, and nucleic acids. They feature a semi-permeable membrane that allows the passage of small molecules while retaining the desired larger molecules.
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Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Exoquick reagent, by System Biosciences (3 mentions)
Exoquick tc exosome isolation reagent, by System Biosciences (3 mentions)
Protein a sepharose fast flow, by GE Healthcare (2 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (2 mentions)
Spin x tube filter, by Corning (2 mentions)
Ni nta agarose, by Qiagen (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Nap 5 column, by GE Healthcare (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
Pd 10 column, by GE Healthcare (2 mentions)
Series 2000, by PerkinElmer (2 mentions)
Exo fbs, by System Biosciences (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Elan drc e, by PerkinElmer (2 mentions)
Clone express 2 one step cloning kit, by Vazyme (2 mentions)
132 protocols using amicon ultra filter
1
Matrigel Plug Angiogenesis Assay
2
Isolation and Preparation of MDM Fractions
3
Saponin-based Nanoparticle Adjuvant Synthesis
4
Tetracycline-Inducible Human ABCD1 Expression
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We utilized the Flp-In TREX system (Thermo Fisher Scientific) to implement tetracycline inducible expression of human ABCD1 to overcome several impediments associated with weak and inconsistent protein yields from transiently transfected HEK293T cells. Briefly, a synthetic gene construct of isoform 1 of human ABCD1 (Uniprot ID 095477) codon optimized for human cell expression (GeneArt/ Scientific) was first cloned in an expression vector comprising the pXLG gene expression cassette in a pUC57 vector backbone (GenScript) between BamHI and SalI restriction sites as described (39) . This added an eYFP-Rho1D4 purification tag preceded by a 3C/precision protease site at the C-terminal end of the construct. The full expression construct of ABCD1 or its EQ variant, generated through site directed mutagenesis using forward primer:
5'-CGGCCTAAGTACGCCCTGCTGGACCAGTGTACAAGCGCCGTGTCCATCG-3' and reverse primer: Ni-NTA Superflow resin in tandem (Qiagen). Eluent was concentrated using 100 kDa cutoff Amicon Ultra filters (Millipore-Sigma).
5'-CGGCCTAAGTACGCCCTGCTGGACCAGTGTACAAGCGCCGTGTCCATCG-3' and reverse primer: Ni-NTA Superflow resin in tandem (Qiagen). Eluent was concentrated using 100 kDa cutoff Amicon Ultra filters (Millipore-Sigma).
5
Quantification of Acrolein in Cell-free Samples
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Cell-free PBS samples were exposed to room air or SHS under same conditions as that of HBE cell exposure with and without 300 μM of NAC, as performed elsewhere [33 ]. Immediately after exposure, PBS samples were processed to generate stable derivatives of acrolein for subsequent estimation by mass spectroscopy. PBS samples were loaded into the 10 kDa MWCO Amicon ultrafilters (Millipore, Billerica, MA) and centrifuged at high speed for 30 min at 4 °C. The filtrate was then derivatized using freshly prepared 2,4-diphenylhydrazine (DNP, Sigma, St Louis, MO) in acetonitrile/formic acid and allowed to react at room temperature for 1 hour. The solution was clarified by centrifugation at high speed for 5 min. Mass spectroscopy was conducted on an API-4000 (AB Sciex, Framingham, MA) equipped with a C18, 100A, 100 × 2.10 Kinetix column (Phenomenex, Torrance, CA). Standard solutions were prepared from acrolein-DNP solid synthesized in-house with > 95% purity by NMR. A gradient of 5–100% solutions containing acetonitrile and 10 mM ammonium acetate) was infused over 10 min at a flow rate of 0.3 ml/min. Analytes were modified with chemical ionization in negative mode. The 235–163 and 235–65 peaks were quantified relative to acrolein standards and the values averaged for each sample, as reported earlier [4 (link)].
6
IgG Digestion and F(ab')2 Purification
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IgG were incubated with IdeS (Dixon, 2014 ) (4 μg of IdeS per 1 mg of IgG) in PBS for 1 h at 37°C. The Fc and IdeS A were removed using a mix of Protein A Sepharose® Fast Flow (250 μL per 1 mg digested mAb; GE Healthcare Life Sciences) and Ni Sepharose™ 6 Fast Flow (50 μL per 1 mg digested mAb; GE Healthcare Life Sciences) which were washed twice with PBS before adding to the reaction mixture. After exactly 10 min the beads were removed from the F(ab’)2-dilution by filtration in Spin-X tube filters (Costar®) and the filtrate was concentrated in Amicon® Ultra Filters (10k, Millipore). Purified F(ab’)2 fragments were analyzed by SDS-PAGE.
7
Viral Genomic DNA Extraction and Purification
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The collected CsCl fractions were dialyzed overnight in autoclaved MilliQ water as described above and then centrifuged in Amicon Ultra filters (Millipore UFC203024) for 15 min at 4,000×g at 15 °C. The volume of each sample was then brought up to 450 μl (using sterile MilliQ water), and 50 μl of 10× DNase I buffer were added to a reach a final volume of 500 μl. To remove external DNA, samples were treated with 2.5 U ml−1 of DNase I and incubated at 37 °C for 1 h. The extraction of virions was carried out as described by Thurber et al. [20 (link)], 0.1 volumes of 2 M Tris HCl/0.2 M EDTA, 5 μl of 0.5 M EDTA (used for inactivation of DNase I), and 1 volume of formamide were added to each sample and incubated at room temperature for 30 min. DNA was spun down by adding 1 volume of 99.9 % ethanol and centrifuging at 14,000×g for 20 min at 4 °C; the pellet was washed twice using 70 % ethanol and re-suspended overnight in 537 μl of TE buffer at 4 °C. Subsequently, 30 μl of 10 % SDS and 3 μl of 20 mg ml−1 proteinase K were added and incubated for 1 h at 55 °C. DNA was washed and concentrated using DNA Clean & Concentrator (Zymo Research D4010) and eluted in 15 μl of elution buffer (the DNA binding buffer to sample ratio was 1:2), and the DNA concentrations were measured using Qubit® (dsDNA HS assay, Invitrogen).
8
Measuring Isocitrate Dehydrogenase Reactions
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Each reaction mixture contained
150 mM NaCl, 2 mM MgCl2, and 20 mM Tris-HCl buffer (pH
7.5 and 7.0). Reduction reaction mixtures contained 8 mM ICT, 200
μM NADP+, and 250 nM total protein (IDH1-WT, IDH1-R132H,
or 1:1 IDH1-WT:IDH1-R132H). Reaction mixtures run in triplicate were
incubated at 24 °C for 1 h; 100 μL reactions were stopped
by boiling for 10 min. Samples were then passed through Amicon Ultra
filters (Millipore) to remove proteins and processed immediately or
flash-frozen and stored at −80 °C. Levels of AKG and D-2HG
were measured using commercial kits (Biovision, D-2HG, catalog no.
K213; AKG, catalog no. K677) and per manufacturer instructions. Four
experimental replicates were analyzed. Per kit recommendations, for
each experimental replicate, two technical replicates were used as
well as two additional technical replicates prepared with a spiked-in
positive control and a background control.
150 mM NaCl, 2 mM MgCl2, and 20 mM Tris-HCl buffer (pH
7.5 and 7.0). Reduction reaction mixtures contained 8 mM ICT, 200
μM NADP+, and 250 nM total protein (IDH1-WT, IDH1-R132H,
or 1:1 IDH1-WT:IDH1-R132H). Reaction mixtures run in triplicate were
incubated at 24 °C for 1 h; 100 μL reactions were stopped
by boiling for 10 min. Samples were then passed through Amicon Ultra
filters (Millipore) to remove proteins and processed immediately or
flash-frozen and stored at −80 °C. Levels of AKG and D-2HG
were measured using commercial kits (Biovision, D-2HG, catalog no.
K213; AKG, catalog no. K677) and per manufacturer instructions. Four
experimental replicates were analyzed. Per kit recommendations, for
each experimental replicate, two technical replicates were used as
well as two additional technical replicates prepared with a spiked-in
positive control and a background control.
9
Recombinant Keratinase Expression in E. coli
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The successful expression construct was transformed into E. coli (DE3) ArticExpress. E. coli was cultured overnight at 37 °C in 20 mL of LB medium containing 50 µg/mL of kanamycin. The cultures were transferred into 1 L of fresh medium and induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside at A600 = 0.6–0.8, followed by overnight expression at 20 °C. The cells were harvested by centrifugation at 6,000 × g for 30 min, resuspended in cold lysis buffer (20 mM imidazole, 250 mM NaCl, and 50 mM HEPES, pH 8.0), and disrupted by French press. The E. coli debris was removed by centrifugation at 8,000 × g for 30 min. The recombinant keratinases were purified by Ni-affinity chromatography (Novagen) and eluted with an increasing imidazole gradient. The eluted keratinase was concentrated and buffer-exchanged using Amicon Ultra filters with a 10-kDa cutoff (Millipore) into keratinase buffer (10 mM CaCl2, 150 mM NaCl, and 50 mM HEPES, pH8.0). The molecular weight of purified rMtaKer was determined by protein electrophoresis and mass spectrometry. The enzyme was protected by United States Patent 9,434,93441 .
10
Inducing AMP Synthesis in Silkworms
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To induce the synthesis of AMPs, B. mori larvae were anesthetized on ice and infected with 10 µL containing 103 CFU of the E. coli/M. luteus suspension in sterile PBS. After infection, larvae were maintained in a climatic chamber for 24 h in the dark, at 25 °C. All experiments were performed under sterile conditions and not infected larvae were used as controls.
Hemolymph plasma was collected from the fifth instar larvae. Larvae were surface sterilized with 70% ethanol and then washed in sterile PBS; anesthetized larvae were bled by puncture of a proleg, and hemolymph flushed out in a refrigerated sterile tube containing a few 1-phenyl-2-thiourea crystals to avoid undesired activation of the prophenoloxidase enzyme. Cell-free plasma was obtained by clarification and increasing centrifugations (up to 1500× g) to remove cells and tissue debris. Whole plasma was filtered on 0.22 µm Minisart or processed by Amicon® Ultrafilters (Millipore, Burlington, MA, USA) to obtain low molecular mass fractions (cut-off 30 kDa). Total protein content was determined by Bradford protein assays calibrated on BSA. All samples were used immediately or stored at −20 °C.
Hemolymph plasma was collected from the fifth instar larvae. Larvae were surface sterilized with 70% ethanol and then washed in sterile PBS; anesthetized larvae were bled by puncture of a proleg, and hemolymph flushed out in a refrigerated sterile tube containing a few 1-phenyl-2-thiourea crystals to avoid undesired activation of the prophenoloxidase enzyme. Cell-free plasma was obtained by clarification and increasing centrifugations (up to 1500× g) to remove cells and tissue debris. Whole plasma was filtered on 0.22 µm Minisart or processed by Amicon® Ultrafilters (Millipore, Burlington, MA, USA) to obtain low molecular mass fractions (cut-off 30 kDa). Total protein content was determined by Bradford protein assays calibrated on BSA. All samples were used immediately or stored at −20 °C.
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