Lipids were purchased from Avanti Polar Lipids, Alabaster, AL. POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) stocks were prepared in chloroform and stored at –20 °C. Lipids were dried under argon gas and stored in a vacuum overnight prior to use in experiments. Lipid films were resuspended as described below. Large unilamellar vesicles (LUVs) were prepared using a Mini-Extruder (Avanti Polar Lipids, Alabaster, AL) with a 100 nm filter (Whatman, United Kingdom) (Nguyen et al., 2015 (link); Scott et al., 2015 (link)).
100 nm filter
Manufactured by Cytiva
Sourced in United Kingdom
The 100-nm filters are a type of laboratory equipment designed to remove particles or contaminants from liquids or gases. They feature a pore size of 100 nanometers, which allows for the filtration of very small substances while maintaining flow rate and throughput. These filters are commonly used in various applications that require high-precision filtration, such as in the biopharmaceutical, food and beverage, and electronics industries.
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Lab products found in correlation
6 protocols using 100 nm filter
1
Preparation of Large Unilamellar Vesicles
2
LUV Preparation and Thrombin Removal
3
Liposome Generation for Biopharma Research
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Liposomes were generated as previously described (Richardson and Fromme, 2015 ). Briefly, individual lipid stocks (Avanti Polar Lipids) were combined in 96.5:2.5:1 DOPC:PIP:DiR-dye molar ratios in the presence of chloroform and methanol. Lipid films were vacuum-dried in pear-shaped flasks and rehydrated overnight to 1 mM at 37°C in HK buffer (20 mM HEPES, pH 7.4, and 150 mM KOAc). Lipids were extruded in a miniextruder (Avanti Polar Lipids) with 100-nm filters (Whatman) using 25 passes through the filter. Liposomes were stored at 4°C and were generally used within 1 wk of extrusion.
4
Synthetic TGN Liposome Preparation
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Synthetic TGN liposomes were prepared using a mixture of lipids that approximates the lipid composition of the TGN (Klemm et al., 2009 (link)). Lipids were combined, vacuum dried, rehydrated in HK buffer (20 mM Hepes, pH 7.4, and 150 mM KOAc), and extruded through 100-nm filters (Whatman) to generate liposomes. See Table S3 for the composition of TGN and phosphatidylcholine liposomes.
5
Liposomal Encapsulation and Release Kinetics
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60 mM pure POPC or a 3:7 ratio of DOPG/POPC liposomes, respectively, with 50 mM CXF in buffer (100 mM KCl, 10 mM Tris, 10 mM MES (2-(N-morpholino)ethanesulfonic acid) (pH 7.0)) were prepared. Solutions were extruded by 100-nm filters (Whatman, Dassel, Germany). The polydispersity of the population was checked and did not exceed 10%. Liposomes were purified by the dialysis method, based on Micro Float-A-Lyzer dialysis membranes (Sigma-Aldrich) with the threshold ∼50 kDa. Lipid concentration was determined using ammonium ferrothiocyanate colorimetric method to assess the loss after ultrafiltration. The final lipid concentration was equal to 20 mM. Kinetics of release of CXF was measured with a Horiba Fluoromax 4 spectrofluorometer (λex 492 nm, slit, 1 nm; λem 517 nm, slit, 1 nm; Horiba, Piscataway, NJ) for ∼700 s with a time step of 1 s. 4 μL of liposome solution was added to 3 mL buffer in measuring cuvette. This corresponds to 27 μM final lipid concentration during the measurement.
6
Synthesis and Characterization of Cationic Liposomes
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DOBAB and MO lipids were purchased from TCI Chemicals (Zwijndrecht, Belgium) and (Sigma-Aldrich San Luis, MO, USA), respectively. DODAB:MO cationic liposomes (molar ratio 1:2) were synthesized by film re-hydration followed by extrusion. Briefly, well-defined volumes of the lipids (20 mM in ethanol) for a final concentration of 3 mM were added to a rounded tube and exposed to vacuum in a bath at 60 °C for 10 min to evaporate the solvent. The formed lipid film was re-hydrated with 5 mL of endotoxin-free water for 15 min at 60 °C under rotation. Finally, to achieve a homogeneous population, the resultant liposomes were extruded 2 and 6 times at 60 °C using 400 and 100 nm filters (Whatman®, Maidstone, UK), respectively. The liposomes were stabilized at room temperature (RT) for 1 h before use.
For cell internalization studies, the nanostructures were labeled with rhodamine B by adding L-α-Phosphatidylethanolamine-N-(lysamine rhodamine B sulfonyl) (Avanti Lipid Polar, Birmingham, AL, USA) (25 μL at 3 mM in absolute ethanol) into the lipid mixture at a molar ratio of 1:200 (rhodamine:lipids) and following the same protocol described for the synthesis of liposomes. To avoid the degradation of the rhodamine, the sample was prepared and stored in dark conditions.
For cell internalization studies, the nanostructures were labeled with rhodamine B by adding L-α-Phosphatidylethanolamine-N-(lysamine rhodamine B sulfonyl) (Avanti Lipid Polar, Birmingham, AL, USA) (25 μL at 3 mM in absolute ethanol) into the lipid mixture at a molar ratio of 1:200 (rhodamine:lipids) and following the same protocol described for the synthesis of liposomes. To avoid the degradation of the rhodamine, the sample was prepared and stored in dark conditions.
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