The xhlAB-xlyA inducible strain, 168 (PxylA-xhlAB-xlyA) or the control strain, 168 (PxylA), was incubated to OD600 of 0.3–0.4 and 0.1% xylose was added. The strains were further incubated for 4 h and then the supernatant was collected. The supernatants were filtered with a PES 0.22 μm filter (Merck Millipore, Ireland). FM4-64FX or FM1-43FX dye fixed late exponential phase B. subtilis 168 was added to the supernatants to examine if MVs can be induced from these cells by exposure to the supernatants. Cells stained with FM4-64FX or FM1-43FX were fixed with 4% paraformaldehyde and the cells were washed twice with PBS after fixation. Heat treatment of the xhlAB-xlyA induced supernatant was carried out at 95 °C for 10 min. The FM dye fixed cells were incubated with the supernatants or MVs at 37 °C for 16 h. After 16 h incubation, the cells were pelleted down and the MVs in the supernatant were isolated by ultracentrifugation as described above. MVs derived from the FM dye fixed cells were quantified by detecting the FM dyes as described for the MV quantification.
0.22 μm pes filter
Manufactured by Merck Group
Sourced in United States
The 0.22 μm PES filter is a laboratory equipment product designed for the filtration of aqueous solutions. It features a polyethersulfone (PES) membrane with a pore size of 0.22 micrometers, which is effective in removing particulates, microorganisms, and other contaminants from liquids. The filter is made to be used in a variety of laboratory applications where high-quality filtration is required.
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Lab products found in correlation
5 protocols using 0.22 μm pes filter
1
Isolation and Quantification of Microvesicles
2
Rat Pulmonary Microvascular Endothelial Cell Infection
3
Conditioned Media Preparation from HSCs
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To prepare conditioned media (CM), HSCs were cultured to reach 80% confluence in 10 cm dishes, and treated as indicated. Then the medium was replaced with fresh complete medium. Alternatively, cyclosporine A (10 μmol/L) was added to HSCs culture for 24 h before replacing the medium with fresh drug-free medium. Forty-eight hours later, the media were collected and filtered through 0.22 μm PES filter (Merck Millipore Ltd., USA) to remove cells and cellular debris. The media were used immediately or aliquoted and stored at −80 °C for later use for culture of HSCs.
4
Preparation of Platelet-Rich Plasma
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Expired platelet units of blood group O and AB, negative for infectious diseases were obtained from the blood banks. Platelet count was enumerated and batch of pHPL with a platelet count of ≥2 × 108 cells/mL was employed in this study. Platelet units were subjected to three freeze-thaw cycles and pooled at the ratio of 3:1 (Blood group O: AB) to counter the effects of ABH antigens and isoagglutinins42 . Pooled platelets were centrifuged at 4000 × g for 15 minutes and the pellet of platelet fragments and cell debris was discarded. Supernatant was strained through a 40μm cell strainer and further filtered through a 0.22 μm PES filter (Merck Millipore, Billerica, MA, USA). Filtered product was stored as aliquots of 10 ml at −80 °C (Fig. 1b ).
5
Microfluidic Synthesis of Lipid Nanoparticles
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LNPs were prepared by microfluidic mixing as described in Belliveau NM et al. [43 (link)]. Briefly, lipids were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid/Phospholipid/cholesterol/PEGLipid). The lipids in ethanol solution at 20 mg/mL and the mRNA in 50 mM citrate buffer (pH 4.0) at 0.265 mg/mL for the preparation of DOG-IM4 and DOG-IM2 LNPs or at 0.305 mg/mL or 0.314 mg/mL for the preparation of respectively L319 LNPs or MC3 LNPs, were then injected into a microfluidic mixer (NanoAssemblr™, Precision Nanosystems, Vancouver, BC) at a flow rate ratio of 1:3 with a combined final flow rate of 4 mL/min. The selected lipid/mRNA concentrations yielded a constant N/P ratio of 6 (cationic nitrogen groups from the ionizable lipid over anionic phosphate groups from the mRNA) corresponding to lipid/mRNA ratios of 25 in DOG-IM4 and DOG-IM2 LNPs or 22 in L319 and MC3 LNPs. Formulations were then dialyzed against 50 mM citrate buffer, pH 4.0, for at least 4 h followed by phosphate buffered saline, pH 7.4 (PBS), for 24 h by using 10 kDa MWCO dialysis cassettes (Spectrum Labs, Rancho Dominguez, CA). Final LNPs were filtered on a 0.22 μm PES filter (Merck-Millipore) and stored liquid at 4 °C in PBS under nitrogen atmosphere.
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