A conditioned medium was used to improve the survival of sorted cells after FACS. To prepare the conditioned medium, BL21 (DE3) cultures were grown in LB (OD600~1.0), centrifuged at 8000 × g for 20 min at 4 °C and the supernatant was sterilized by passing through a 0.22 μm cellulose filter (Millipore, Bedford, MA).
0.22 μm cellulose filter
Manufactured by Merck Group
Sourced in United Kingdom, India
The 0.22 μm cellulose filter is a laboratory equipment used for the filtration of liquids. It has a pore size of 0.22 micrometers, which allows the effective removal of bacteria and other microorganisms from the filtered solution.
Automatically generated - may contain errors
4 protocols using 0.22 μm cellulose filter
1
Conditioned Medium for Sorted Cell Survival
2
Integrated Hybrid Bioreactor Design
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A three-chambered integrated hybrid bioreactor was designed by combining the plasmapheresis module and adsorbent module together with the bioreactor module. Three disc shaped units were placed such that the top unit consists of a plasma separation membrane (VividTM Plasma Separation Membrane, Pall Life Sciences, USA) placed over an activated charcoal cloth (a gift from Prof. Nishith Verma, Department of Chemical Engineering, Indian Institute of Technology Kanpur, India); the middle unit contains the hepatocyte seeded cryogel disc; and the lower unit contains a 0.22 μm cellulose filter (Millipore, India). Each chamber is connected to the other and has an inlet and outlet to allow for exchange of nutrients as well as unhindered circulation of blood and plasma. The total volume of the entire system is approximately 4 mL (Fig. 1A–C ).
3
Fluorescence-based Peptide Transport Assay
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Transport assays were carried out as described previously (22 (link)), employing a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) to measure the change in fluorescence of the pH-sensitive dye pyranine. Dual fluorescence excitation was set to 460/415 nm with emission at 510 nm. Transport was initiated following addition of 1 μM valinomycin. Competition assays were performed at 30 °C, with samples taken at specified time points. Proteoliposomes were immediately filtered onto a 0.22-μm cellulose filter (Merck Millipore) using a vacuum manifold and washed twice with 2 mL cold H2O. The amount of peptide transported into the liposomes was calculated based on the specific activity for each peptide. Experiments were performed a minimal of four times to generate an overall mean and SD and the resulting data were analyzed using Prism 7.0 (GraphPad Software).
4
Proteoliposome Transport Assay
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Proteoliposomes were harvested as before, the resulting pellet was resuspended in INSIDE buffer, subjected to three freeze-thaw cycles in liquid nitrogen, extruded through a 0.4 μm polycarbonate membrane and harvested by ultracentrifugation once more. For the assay, proteoliposomes were diluted in external buffer (110 mM NaCl, 10 mM NaPO4 and 2 mM MgSO4) supplemented with 50 μM di-alanine peptide containing trace amounts of tritiated 3H-di-alanine (specific activity 30 Ci/mmol) and the competing substrate at increasing concentrations as indicated. Competition assays were performed at 30°C, samples were taken at specified time points and transport was stopped by addition of 2 mL H2O. Proteoliposomes were immediately filtered onto a 0.22 μm cellulose filter (Merck Millipore) using a vacuum manifold and subsequently washed twice more with 2 mL H2O. The amount of peptide transported into the liposomes was calculated based on the specific activity for each peptide as detailed by the manufacturer and counting efficiency for the radioisotope in Ultima Gold (PerkinElmer) counted in a Wallac scintillation counter (3H, 45% counting efficiency). Transport assays were performed a minimum of three times to generate an overall mean and S.E.M.
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