AF4 measurements were carried out with the ‘AF2000 MT Series mid temperature’ (Postnova Analytics) system equipped with a 350 µm channel and a cellulose membrane (cut-off: 5 kDa; Postnova Analytics). The channel was constantly maintained at 40°C. Filtered 95% ethanol (0.1 µm Millipore filter disc) was used as flowing liquid for the AF4. The channel flows were controlled by ‘AF2000 Control Program’ software (Postnova Analytics). Samples were injected into the channel at a flow of 0.1 ml min−1 with a focus flow of 3.1 ml min−1. During the injection time of 10 min and an additional transition time of 1 min, the cross-flow was kept constant at 2.5 ml min−1. After transition (i.e. deletion of the focus flow) the cross-flow was reduced within 10 min to 0.1 ml min−1 (power gradient of 0.2), followed by a linear decline during 10 min until the separation force reached zero. The main channel flow was kept constant at 0.7 ml min−1 during the entire run. Samples were injected by full-loop injections of a PN5300 series autosampler (Postnova Analytics) equipped with a 1000 µl sample loop.
Millipore filter disc
Manufactured by Merck Group
The Millipore filter disc is a laboratory filtration device. It is designed to filter liquids and remove particulates or contaminants from the sample. The filter disc is made of a porous material that allows the liquid to pass through while trapping the desired substances.
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3 protocols using millipore filter disc
1
Asymmetrical Flow Field-Flow Fractionation
2
Molar Enamel Development under EZH2 Inhibition
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Tooth organs from e16 embryos (obtained from C57BL/6 timed-pregnant females, Charles River) were dissected and late cap stage M1 molars were isolated. The organs were grown for 10 days in a Trowell Organ culture system using 1X BGJb medium (Fitton-Jackson, Gibco), 20% FBS, 100 µg/mL ascorbic acid, 100 U/mL penicillin, and 100 µg/mL streptomycin (Evans et al. 1998). The molar explants were oriented on a Millipore filter disc to identify left and right quadrant tooth organs. EZH2 inhibition was carried out by adding the selective inhibitor GSK126 (5 µM final concentration). Enamel thickness measurements were carried out using Image J software (NIH, v1.52a). A minimum of 50 measurements were performed for each experimental condition.
3
Antifungal Effects of Garlic and Onion Extracts
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This experiment aimed to study the effect of garlic and onion extracts on the mycelial growth of the tested fungi. Materials were crushing in blender, then squeezed twice through eight layers of cheese cloth. The extract was filtered through a double cheesecloth fabric and Whatman No.1 filter paper and centrifuged for 10 minutes at 300 rpm to get a clear plant extract. Sterilization was made using 0.45 µm Millipore filter disc. The crude extract was reduced with sterile distilled water to give a series of concentrations of its original volume treatment. (Shalaby, 1993) . Two concentrations; i.e.25 and 50% of garlic and onion extracts were prepared and poured in, melted PDA flask 250 ml just before solidification (40C 0 ) and the poisoned melted media were poured in sterilized Petri dishes plates 9 cm which were inoculated with equal disks (4mm in diameter) of the desired fungus, taken from 7 dayold cultures. Four replicates were used for each treatment. The control was carried out without addition of any plant extract. The plates were incubated at 25 ± 2 C 0 . The diameter of the mycelial growth in all treatments was recorded, when the mycelial growth covered the plate surface of the control of each fungus.
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