OVA was dialyzed for 36 h at 4°C with several changes of the buffer (PBS) to remove degradation products as well as possible contaminating peptides from the solution. Small peptides, PL19 (PDEVSGLEQLESIINFEKL) were loaded onto Hsp90 in solution at a molar ratio of 50:1 in PBS, incubated for 10 min at 45–50°C, and cooled at room temperature for 30–40 min. For Hsp90–OVA conjugate preparation, soluble Hsp90 and excess OVA (1:2 ratio) were mixed and incubated for 10 min at 45°C. The solution mixtures were then incubated at room temperature for 30 min. Free OVA was removed using Microcon YM-100 (Millipore, Bedford, MA) with a 100-kDa cutoff. Alexa 488- and Alexa 555-labeled Hsp90-peptide/OVA (Hsp90.PC) conjugates were made according to the manufacturer’s protocol (Invitrogen). For anti–SREC-I Ab-OVA preparation, OVA was coupled to anti–SREC-I Ab using bis-(sulfosuccinimidyl) suberate as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA) and labeled with Alexa fluorochromes as described for Hsp90 and purified using Microcon YM-100 ultrafiltration (Millipore, Billerica, MA).
Microcon ym 100
Manufactured by Merck Group
Sourced in United States
The Microcon YM-100 is a centrifugal filter device designed for the concentration and desalting of macromolecular solutions. It utilizes a regenerated cellulose membrane with a molecular weight cutoff of 100,000 Daltons to separate and concentrate sample components.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Pkh67 green fluorescent labelling kit, by Merck Group (2 mentions)
Bis sulfosuccinimidyl suberate, by Thermo Fisher Scientific (1 mentions)
Pkh26 dye, by Merck Group (1 mentions)
Eclipse ti s fluorescence microscope, by Nikon (1 mentions)
Pkh67 green fluorescent labeling kit, by Merck Group (1 mentions)
Mmessage mmachine kit, by Thermo Fisher Scientific (1 mentions)
Genechip human genome u133a 2.0 array, by Thermo Fisher Scientific (1 mentions)
Genome analyzer iix, by Illumina (1 mentions)
Repli g mini kit, by Qiagen (1 mentions)
Nebnext dna library prep master mix set, by Illumina (1 mentions)
Avance 600 spectrometer, by Bruker (1 mentions)
Esi source, by Thermo Fisher Scientific (1 mentions)
Lcq fleet ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Av 400, by Bruker (1 mentions)
Ultraviolet visible spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Bs124s electronic balance, by Sartorius (1 mentions)
12 well plate, by Corning (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Pkh67 green fluorescence labelling kit, by Merck Group (1 mentions)
21 protocols using microcon ym 100
1
Hsp90-peptide/OVA conjugate preparation
2
EV Labeling and Cellular Uptake
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We utilized the PKH26 dye (Sigma Aldrich, USA) to label collected EVs, which were then passed through a 10-kDa filter (Microcon YM-100, Millipore, USA) and washed thrice with PBS. These labeled EVs were then combined overnight with 786-O and Caki-1 cells in serum-free medium at 37°C. Cells were then fixed using 4% paraformaldehyde, stained using 4', 6-diamidino-2-phenylindole (DAPI; Sigma), and analyzed with an Eclipse Ti-S fluorescence microscope (Nikon, Japan).
3
Labeling and Uptake of Extracellular Vesicles
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Purified EVs derived from MG63 cells were labeled with the PKH67 Green Fluorescent Labeling Kit (Sigma‐Aldrich, St. Louis, MO, USA). EVs were incubated with 2 μm PKH67 for 5 min, washed four times through a 100‐kD filter (Microcon YM‐100; Millipore) to remove excess dye, and incubated with C4‐2B cells at 37 °C.
4
Labeling and Uptake of Extracellular Vesicles
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Purified EVs derived from PC3M and PNT2 cells were labelled with the PKH67 Green Fluorescent Labelling Kit (Sigma‒Aldrich). EVs were incubated with 2 μM PKH67 for 5 min, washed four times through a 100‐kDa filter (Microcon YM‐100, Millipore) to remove excess dye, and incubated with RAW264.7 cells in the presence of RANKL (10 ng/mL) at 37°C.
5
Separating Platelet Aggregation Mediators
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To estimate the relative size of the soluble HW-derived mediators involved in platelet
aggregation, the HW extract was separated with a molecular weight (MW) limit of 100,000 by
ultrafiltration using a Microcon YM-100 centrifugal filter device (Millipore, Billerica,
MA, U.S.A.). Briefly, 100 µl of the HW extract was added to the device
and centrifuged at 500 ×g for 15 min at room temperature. After
centrifugation, the filtrated liquids (including substances with a MW<100,000) and the
remaining liquids (including substances with a MW≥100,000) were collected and subjected to
a platelet aggregation assay, respectively.
aggregation, the HW extract was separated with a molecular weight (MW) limit of 100,000 by
ultrafiltration using a Microcon YM-100 centrifugal filter device (Millipore, Billerica,
MA, U.S.A.). Briefly, 100 µl of the HW extract was added to the device
and centrifuged at 500 ×g for 15 min at room temperature. After
centrifugation, the filtrated liquids (including substances with a MW<100,000) and the
remaining liquids (including substances with a MW≥100,000) were collected and subjected to
a platelet aggregation assay, respectively.
6
Synthesizing and Injecting mRNA and Morpholinos in Zebrafish
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mRNAs were synthesized from NotI digested pCS2+hHDAC1 wt, S406A, S406E plasmids using mMessage mMachine kit (Ambion) and purified with Microcon YM-100 (Millipore) filter devices. RNA quality was assayed by means of gel electrophoresis. mRNAs was then diluted in 1X Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6) at final concentration of 120 ng/μl and pressure injected into 1–2 cell stage embryos.
Morpholinos were purchased by Gene Tools, LLC. The morpholino against hdac1 (5′-TTGTTCCTTGAGAACTCAGCGCCAT-3′) was targeted to the translational initiation site, as ref. 19 (link). The scramble morpholino sequences was: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. Morpholinos (0,15 mM) were diluted and injected as described above.
Morpholinos were purchased by Gene Tools, LLC. The morpholino against hdac1 (5′-TTGTTCCTTGAGAACTCAGCGCCAT-3′) was targeted to the translational initiation site, as ref. 19 (link). The scramble morpholino sequences was: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. Morpholinos (0,15 mM) were diluted and injected as described above.
7
miRNA Expression Profiling Protocol
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miR expression was analyzed using miR expression profiling (LC Sciences, TX). 5 μg of total RNA for each sample was fractionated using the Microcon YM-100 (Millipore, MA), and the small RNAs (< 300 nucleotides) were 3′-extended with a poly(A) tail by poly(A) polymerase. miR expression profiling was carried out as previously described [18 (link)]. mRNA expression levels for use in miR activity analysis were determined using GeneChip Human Genome U133A 2.0 Array (Affymetrix, CA) performed at the MGH Core Facility.
8
Amplification and Sequencing of eccDNA
9
Purification of Human SNF2h and hACF
10
Analytical Techniques for Chemical Characterization
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A BS-124S electronic balance (Sartorius, Gottingen, Germany) was used for weight measurements. An ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for absorbance measurements. HPLC was conducted on a Waters 2695 instrument (DAD, Milford, CT, USA) coupled to a Waters 2998 diode array detector (DAD). A Sigma 1–14 centrifuge (Sigma Zentrifugen, Osterode am Harz, Germany) equipped with an ultra-membrane filter Microcon YM–100 (Millipore, Bedford, MA, USA) was also used. ESI–MS was performed using an LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an ESI source (Thermo Fisher Scientific, San Jose, CA, USA). HSCCC was performed on a TBE-300B Spectrum HSCCC (Shanghai Tauto Biotech Co., Ltd., Shanghai, China). 1H and 13C NMR spectra were recorded on a Bruker AV-400 and Avance-600 spectrometer (Bruker BioSpin, Ettlingen, Germany).
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