The NPN assay was performed as described previously17 (link). Overnight cultures were diluted and grown to OD600 = 0.6-0.8, and then normalized to OD600 = 0.5. Cells were washed and resuspended in 5 mM HEPES buffer (pH 7.2). Two hundred microliters of cells were aliquoted into a clear-bottom black-well plate (Corning Incorporated, Cat. #3603). NPN was added at a final concentration of 10 μM. The plate was sealed with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich, Cat. #Z380059) and incubated in a Synergy H1 microplate reader (BioTek). Fluorescence (excitation 350 nm, emission 420 nm) was measured after 20 min of incubation.
Breathe easy gas permeable membrane
Manufactured by Merck Group
Sourced in United States
The Breathe-Easy gas-permeable membrane is a laboratory equipment product designed for controlled gas exchange. It allows for the transfer of gases, such as oxygen and carbon dioxide, while maintaining a barrier to liquids and other materials. The membrane is engineered to be gas-permeable, facilitating the passage of specific gases as per the requirements of the laboratory application.
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Lab products found in correlation
6 protocols using breathe easy gas permeable membrane
1
NPN Assay for Membrane Permeability
2
Growth Kinetics Monitoring of Bacterial Cultures
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Overnight cultures were diluted 1:100 in fresh LB and grown at 30 °C for 2 h.
Optical density was measured, and cells were diluted to OD600=0.05 in 500 mL of LB in a 24-well plate (CytoOne, USA Scientific, cat# CC7682-7524). The plate was covered with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich, Cat. #Z380059) and grown overnight in a Synergy H1 microplate reader (BioTek) at 37 °C with continuous orbital shaking at 559 cpm frequency. OD600 was measured every 15 min for 18 h.
Optical density was measured, and cells were diluted to OD600=0.05 in 500 mL of LB in a 24-well plate (CytoOne, USA Scientific, cat# CC7682-7524). The plate was covered with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich, Cat. #Z380059) and grown overnight in a Synergy H1 microplate reader (BioTek) at 37 °C with continuous orbital shaking at 559 cpm frequency. OD600 was measured every 15 min for 18 h.
3
Spent Media Protocol for Cell Lysis
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To make spent media, the wild-type MC4100 strain was grown in LB medium overnight, followed by centrifugation and filtration through a 0.2 μm filter to create cell-free spent medium. Early exponential phase cells (OD600= 0.6–0.8) were concentrated to OD600=1 via centrifugation. Supernatant was discarded and the cell pellet was resuspended in 1 mL of spent medium, transferred to a 24-well plate (CytoOne, USA Scientific, Cat .#CC7682-7524), and covered with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich, Cat. #Z380059). Lysis was monitored by decrease in optical density in a Synergy H1 microplate reader (BioTek) at 37°C with continuous orbital shaking at 559 cpm frequency. OD600 was measured every 15 min for 18 h.
4
NPN Assay for Membrane Permeability
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The NPN assay was performed as described previously (17 (link)). Overnight cultures were diluted and grown to OD600=0.6–0.8, and then normalized to OD600=0.5. Cells were washed and resuspended in 5 mM HEPES buffer (pH 7.2). Two hundred microliters of cells were aliquoted into a clear-bottom black-well plate (Corning Incorporated, Cat. #3603). NPN was added at a final concentration of 10 μM. The plate was sealed with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich, Cat. #Z380059) and incubated in a Synergy H1 microplate reader (BioTek). Fluorescence (excitation 350 nm, emission 420 nm) was measured after 20 min of incubation.
5
Bacterial Growth Kinetics Assay
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Overnight cultures were diluted 1:100 in fresh LB and grown at 30 °C for 2 h. Optical density was measured, and cells were diluted to OD600 = 0.05 in 500 mL of LB in a 24-well plate (CytoOne, USA Scientific, Cat. #CC7682-7524). The plate was covered with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich, Cat. #Z380059) and grown overnight in a Synergy H1 microplate reader (BioTek) at 37 °C with continuous orbital shaking at 559 cpm frequency. OD600 was measured every 15 min for 18 h.
6
Measuring Thiamin Uptake in Yeast Cells
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200 μL SD-Thiamin and 200 μL SD-Thiamin supplemented with 10 μM thiamin were added to triplicate wells in a round-bottom 96 well plate. 10 μL cells (A600 of 2) was added to each and the plate was covered with a Breathe-Easy gas-permeable membrane (Sigma-Aldrich). This was placed in a Synergy plate reader and maintained at 30°C. The A600 and fluorescence were measured at time zero, then every 20 min for 24 h with vigorous shaking. For fluorescence, excitation was measured using 590/20 nm filters, where 590 nm denotes the arithmetic mean of the wavelength at 50% of peak transmission, and 20 nm denotes the full-width at half the maximum (FWHM) transmission, which is the bandwidth at 50% of peak transmission. Emission was measured with 645/40 nm filters. The mean of the media-only wells was subtracted from each replicate. The error bars show the standard deviation calculated from the error of propagation (s(x/y) = (x/y)(sqrt(sumsq(s(i)/m(i))))).
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