Pituitaries removed from transgenic rats, were washed in medium (DMEM without phenol red) and sliced to 250μm thickness in the coronal orientation using a vibrating microtome (Campden Instruments, UK). Pituitary slices were placed on top of a Millicell (Merck Millipore, UK) 0.4μm sterilised culture plate insert filter, positioned in a 35mm glass-coverslip-based dish (Greiner Bio-One, UK) containing 1.3 ml primary cell culture media (DMEM supplemented with 4.5 g/l glucose, 10% Dextran-Charcoal treated FBS (Perbio Scientific, UK), 50 μM Sodium Pyruvate, 0.1μM Ultraglutamine and 500U Penicillin/ Streptomycin. Plates were sealed with a Breathe-Easy® air permeable membrane (Sigma-Aldrich, UK).
Breathe easy
Manufactured by Merck Group
Sourced in United Kingdom, Israel
Breathe-Easy is a compact and portable lab equipment designed to facilitate respiratory monitoring and analysis. It provides accurate measurements of various respiratory parameters, enabling researchers and clinicians to assess lung function and respiratory health.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Corning (4 mentions)
Elx808 absorbance microplate reader, by Agilent Technologies (3 mentions)
Elx808 plate reader, by Agilent Technologies (2 mentions)
96 well clear flat bottom microplate, by Corning (2 mentions)
Infinite m200 pro, by Tecan (2 mentions)
F 45 12 11 usa rotor, by Eppendorf (2 mentions)
Synergy h1, by Agilent Technologies (2 mentions)
Sola light engine, by Lumencor (2 mentions)
Vibrating microtome, by Campden Instruments (1 mentions)
Millicell, by Merck Group (1 mentions)
Synergy 2 reader, by Agilent Technologies (1 mentions)
96 well microtiter plate, by Sarstedt (1 mentions)
Powerwave xs2, by Agilent Technologies (1 mentions)
Elx808, by Agilent Technologies (1 mentions)
Trehalose, by Merck Group (1 mentions)
Y1375, by Merck Group (1 mentions)
Stripwell microplates, by Corning (1 mentions)
384 well plate, by Greiner (1 mentions)
Clear bottom 96 well plate, by Corning (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
28 protocols using breathe easy
1
Pituitary Slice Culture Protocol
2
Measuring Bacterial Growth Kinetics
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The growth rate of each functional clone was measured as the maximum increase in OD over time during exponential growth. Individual colonies were picked and placed onto a pre-inoculation plate and grown for 2–3 h with shaking at 37 °C before inoculation of the final measurement plate. Breathe-Easy (Sigma-Aldrich) film was applied to minimize evaporation during measurements. OD measurements were conducted in 96-well plates containing 150 µL LB medium per well by the ELx808 plate reader (BioTek, USA). OD at 600 nms was measured over 5-min intervals for a maximum of 16 h, and the plates were incubated at the medium shaking setting at 37 °C between measurements.
3
Glycerol-Based Bacterial Growth Kinetics
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All evolved lines and the ancestor were revived from freezer stocks into 2 ml LB and incubated for 12 h with shaking at 250 rpm at 37°C. The cultures were then sub-cultured 1:100 in 2 ml M9 medium, containing 0.2% glycerol as the carbon source, for 12 h. From each tube, cells were then sub-cultured 1:100 in 2 ml M9 glycerol media (a) with and (b) without 0.2% rhamnose, and allowed to grow for 12 h at 250 rpm at 37°C. After growth for 12 h, all lines were sub-cultured to the same initial OD (0.1) into 2 ml M9 glycerol with PQ (40 μM). A volume of 150 μL of these cultures were transferred to a 96-well clear flat-bottom microplate (Costar) in triplicates. The cultures were grown at 37°C in a microplate reader (Tecan Infinite M200 Pro), until they reached stationary phase. OD600 readings were taken every 30 min with 10 min of orbital shaking at 5 mm amplitude before the readings. A gas permeable Breathe-Easy (Sigma-Aldrich) sealing membrane was used to seal the 96-well plates. Growth rate (Malthusian parameter, r) was calculated from the time to reach an OD 1.0, assuming exponential growth in this duration. This growth rate is presented in the growth rates in Figures 4D , 5 .
4
Growth Kinetics of Bacterial Strains
5
Functional STNhaA production assay
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Production of functional STNhaA was probed with a growth complementation assay using the Na+/H+-antiporter deficient E. coli strain EP432 (melBLid, ΔnhaA1::kan, ΔnhaB1::cat, ΔlacZY, thr1 [30] (link). EP432 cells were transformed either with pTTQ18A1-STNhaA-His6 or with pTTQ18A1 vector. Pre-cultures in LBK were inoculated with a single colony. Aliquots of the pre-culture were diluted to an OD600 nm of 0.02 in buffered (50 mM HEPES pH 7.5 or Tris/HCl pH 8.0) LB-medium (10 g/l bactotryptone, 5 g/l yeast extract) complemented with either 200 mM NaCl, 800 mM NaCl or 200 mM LiCl. 100 µl aliquots of the diluted suspension were applied to each well of a 96-well microtiterplate, which was sealed with an adhesive membrane (Breathe-Easy, Sigma-Aldrich). Growth was monitored every 10 min at 600 nm for up to 22 h using a microplate reader (Powerwave XS2, Biotek) at 37°C and under continues shaking. Assays were performed in triplicates in at least three independent experiments.
6
Thioflavin T Protein Aggregation Assay
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100 μL samples containing 100 μM thioflavin T and 50 μM protein in 200 mM ammonium acetate, pH 6.8 and a 1 % (v/v) final concentration of DMSO were prepared in a 96-well plate (CoStar) and sealed with clear sealing film (Breathe Easy, Sigma). Plates were incubated in a FLUOstar OPTIMA plate reader for 5 days at 25 °C without agitation. Fluorescence was excited using a 440 ± 5 nm filter, and emission intensity was measured using a 485 ± 5 nm filter.
7
Bacterial Growth Kinetics Measurement
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Growth rate was measured as the maximum increase in optical density (OD) over time during exponential growth. OD measurements were conducted in 96-well plates containing 150 µl LB/well by the ELx808 plate reader (BioTek, USA). Breathe-Easy (Sigma-Aldrich) film was applied to minimize evaporation during measurements. OD at 600 nms was measured with 5 min intervals for maximum 16 h and incubated with medium shaking at 37°C between measurements.
Colonies were picked into a pre-inoculation plate and were grown for 2–3 h with shaking at 37°C before inoculation of the final measurement plate.
Colonies were picked into a pre-inoculation plate and were grown for 2–3 h with shaking at 37°C before inoculation of the final measurement plate.
8
Curcumin Inhibits Enzyme Activity
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βla-linker or βla-hIAPP (in 50 mM sodium phosphate buffer, pH 7) was added to wells of a 96-well plate (CoStar) to give a final enzyme concentration of 32 μM. 3.2 μL of 10 mM curcumin (in 100 % DMSO) was added to give a final small molecule concentration of 320 μM. For assays in the absence of curcumin, 3.2 μL of DMSO was added instead. Solutions were made up to 100 μL with 50 mM sodium phosphate buffer, pH 7 and the plates sealed with transparent, hydrophobic and gas permeable plastic films (Breathe Easy, Sigma). Plates were incubated for 5 days (quiescent, 25 °C). After 5 days, the enzyme activity of the whole sample was measured using a final enzyme concentration of 0.01 pmol/μL as described above. The samples were then centrifuged (1 h, 16,250 rpm, F-45-12-11 Eppendorf USA rotor) and the concentration of protein remaining in solution determined by the absorbance at 280 nm and the molar extinction coefficient of βla-linker (28,085 M−1 cm−1) or βla-hIAPP (29,700 M−1 cm−1) (samples were base-line corrected using 100 μL of 50 mM sodium phosphate buffer, pH 7, containing a final concentration of 320 μM curcumin, or an equivalent volume of DMSO). The enzyme assay was repeated and the amount of nitrocefin hydrolyzed per min mg of protein was calculated. The specific activity was calculated as a percentage of the initial specific activity (activity at t = 0 was 100 %).
9
Bacterial Growth and Luminescence Assay
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The assays were conducted in 96-well plates. The wells were filled with 200 μL of 0.4 % glucose M63 minimal media containing increasing concentrations of metals, inoculated with 106 bacteria harvested at OD600 of 0.6. The plate was sealed using gas-permeable Breathe Easy membrane (Sigma Aldrich) and placed into a “TECAN Infinite Pro” plate reader equilibrated at 37 °C and programmed to measure OD600 and luminescence every 20 min, after a 1-min period of shaking, during 12–20 h. Background OD and luminescence (values at time = 0) were subtracted to each data point. To calculate the maximal activity, for each well, the ratio (luminescence/OD) was plotted as a function of time, and the maximal value was conserved. By doing so, we took into account the eventual growth lag between two conditions.
10
Air-Drying Budding Yeast for Spaceflight
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The method to air-dry budding yeast cells for the BioSentinel mission has been described previously (Santa Maria et al., 2020 (link)). Briefly, yeast from frozen glycerol stocks were grown on yeast extract-peptone-dextrose (YPD) agar (Y1500; Sigma-Aldrich) for 2–3 days at 30°C. Samples from freshly grown patches were then inoculated into 5 mL of liquid YPD (Y1375; Sigma-Aldrich) and grown on a rotating mixer at ambient temperature (∼23°C) for 7 days. Liquid cultures were pelleted and washed with sterile water, and the cell density was determined by hemacytometer counting. Cell suspensions were prepared to a final density of 1 × 107 cells in 1 mL of 10% trehalose (T0167; Sigma-Aldrich). Ten microliter aliquots of the 1 × 107 cell suspension (containing ∼105 cells) were gently dispensed into the bottom edge of the wells of 96-well Stripwell™ microplates (9102; Costar®) or onto the sidewall of each microfluidic card well (Padgen et al., 2021 (link)).
Plates were sealed with Breathe-Easy® gas permeable sealing membrane (Z380059; Sigma-Aldrich), and the cells were air dried in a 23°C incubator, 20–30% relative humidity, for 7 days. Yeast cells in fluidic cards were air dried inside sterile boxes at the same conditions before card sealing using a pneumatic press (Padgen et al., 2021 (link)).
Plates were sealed with Breathe-Easy® gas permeable sealing membrane (Z380059; Sigma-Aldrich), and the cells were air dried in a 23°C incubator, 20–30% relative humidity, for 7 days. Yeast cells in fluidic cards were air dried inside sterile boxes at the same conditions before card sealing using a pneumatic press (Padgen et al., 2021 (link)).
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