Extraction of volatiles from plant tissue was carried out with 4 g of dry rosemary leaves from varieties '2' and '11' collected from the plant living germplasm collection at Newe Ya'ar Research Center. The vegetative materials were stored in 0.02 L glass sealed vials containing 10-ppm tert methyl butyl ether (MTBE) (99.8%) (BIO-LAB, Israel), with an internal standard of isobutylbenzene (IBB) (Aldrich, Israel CAS Number 538-93-2). The extraction ratio was 1 g vegetative material to 0.01 L solvent volume. The samples were shaken for 24 h at room temperature, and then transferred through a Pasteur pipette containing anhydrous sodium sulfate (Merck) and silica gel 60 (230–400 mesh) (Merck) for cleaning, drying and filtering polar components with high molecular weight (e.g. chlorophyll). The cleaned extracted samples were collected in 0.002 L glass bottles, closed with Teflon covers and stored at 4°C before injection into the GC-MS for analysis [41 ]. GC-MS analysis of the 0.002 L solvent extraction product achieved additional verification of the rosemary components [42 (link)].
Pasteur pipette
Manufactured by Merck Group
A Pasteur pipette is a type of glass or plastic pipette used in laboratory settings. It is a narrow, straight tube with a bulb at the top, used to accurately transfer small volumes of liquid. The Pasteur pipette functions as a manual liquid transfer device, allowing for precise and controlled dispensing of liquids.
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Lab products found in correlation
Silica gel 60, by Merck Group (2 mentions)
Anhydrous sodium sulfate, by Merck Group (1 mentions)
Htf medium, by Merck Group (1 mentions)
M2 media, by Merck Group (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Laminin, by Merck Group (1 mentions)
Neurobasal a, by Thermo Fisher Scientific (1 mentions)
Poly ornithine, by Merck Group (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Anhydrous sodium sulphate, by Merck Group (1 mentions)
Zb 5ms column, by Phenomenex (1 mentions)
α bisabolol, by Merck Group (1 mentions)
5795c mass selective triple axis detector, by Agilent Technologies (1 mentions)
Agilent 7890a, by Agilent Technologies (1 mentions)
Attofluor chamber, by Thermo Fisher Scientific (1 mentions)
Spinning disc confocal microscope, by Olympus (1 mentions)
6 protocols using pasteur pipette
1
Rosemary Volatile Extraction and Characterization
2
Chromatographic Extraction of Epicuticular Waxes
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The concentrated extracts, obtained as described above, were chromatographed on small-scale columns using a Pasteur pipette filled with silica gel 60 (SiO 2 , 0.2 -0.5 mm; Merck) previously activated at 120°C (Mimura et al. 1998) . The wax samples were obtained by elution with 5 ml of hexane and stored at -20°C until further analysis. Organic solvents like benzene, chloroform, hexane, acetone, dichloromethane, methanol, and ethanol have been used on a large scale for surface wax extraction. Hexane is most commonly used in extraction nonpolar fraction of epicuticular waxes including n-alkanes (chain-length C21-C35) and primary alcohols (C22-C40). There are numerous previous reports efficiency of this method (Erosa et al. 2002; Medina et al. 2006; Nikolić et al. 2010; Kundu and Sinhababu 2013; Mitić et al. 2018b, etc.) .
For this both nonacosan-10-ol and n-alkanes analyses one-, and two-year and sometimes threeyear -old needles of trees, collected in spring and autumn separately (as a bulk) were used (ca. four to six repetitions of every taxon).
For this both nonacosan-10-ol and n-alkanes analyses one-, and two-year and sometimes threeyear -old needles of trees, collected in spring and autumn separately (as a bulk) were used (ca. four to six repetitions of every taxon).
3
Melatonin and MPO Effects on Oocyte Quality
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Cumulus oocytes retrieved from the oviductal ampullae were treated with 0.1% hyaluronidase (w/v) in Human tubular fluid (HTF) media (Sigma–Aldrich (St. Louis, MO, USA)) for 2–4 minutes at 37°C. Oocytes were subsequently denuded to remove all cumulus cells with a narrow bore pulled glass Pasteur pipette, thoroughly rinsed in M2 media (Sigma–Aldrich), then the oocytes screened for the presence of the polar body confirming their Metaphase II stage. Oocytes then kept in HTF medium (Sigma–Aldrich) pre-equilibrated with 5% CO2 in air at 37°C in a common pool before randomly transferred into test and control groups. Twenty non cumulus oocytes were used for H2O2 electrode experiment.
Non cumulus oocytes were pre-incubated with 100 μM melatonin in HTF media and treated with 40 nM MPO for 24 h. Of note, HTF media contains Cl- levels akin to the oviductal fluid (~100mM). Treated oocytes were then subjected to indirect fluorescence immunocytochemistry to assess the alterations in metaphase-II mouse oocyte microtubules morphology (MT) and chromosomal alignment (CH) (markers of oocytes quality), and compared to untreated oocytes and oocytes incubated with melatonin (100 μM) alone for 24 h. In the same experiment, all the HTF media from the all treated and untreated groups was filtered and investigated using HPLC.
Non cumulus oocytes were pre-incubated with 100 μM melatonin in HTF media and treated with 40 nM MPO for 24 h. Of note, HTF media contains Cl- levels akin to the oviductal fluid (~100mM). Treated oocytes were then subjected to indirect fluorescence immunocytochemistry to assess the alterations in metaphase-II mouse oocyte microtubules morphology (MT) and chromosomal alignment (CH) (markers of oocytes quality), and compared to untreated oocytes and oocytes incubated with melatonin (100 μM) alone for 24 h. In the same experiment, all the HTF media from the all treated and untreated groups was filtered and investigated using HPLC.
4
DRG Neuron Isolation and Culture
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DRG neurons were harvested from mice and enzymatically dissociated in HBSS containing 2 mg/mL collagenase type I and 4 mg/mL dispase (both from Invitrogen) for 45 minutes at 37°C. DRGs were rinsed twice in HBSS and once in Neurobasal A culture medium (Thermo Fisher Scientific) supplemented with 2% B-27, 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, 50 ng/mL nerve growth factor (NGF), and 50 ng/mL glial cell–derived neurotrophic factor (GDNF) (all from Invitrogen). Individual neurons were dispersed by trituration through a fire-polished glass Pasteur pipette in 4 mL medium and cultured overnight at 37°C with 5% CO2 in 95% humidity on glass coverslips previously treated with poly-ornithine and laminin (both from Sigma-Aldrich).
5
Sesquiterpene Quantification by GC-MS
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Sesquiterpene quantification was performed with GC‐MS. The chloroform: methanol: acetone 2:1:0.5 (v/v/v) extracts were dried by passing them through a Pasteur pipette filled with anhydrous sodium sulphate (Sigma‐Aldrich). Samples were analysed on an Agilent 7890A gas chromatograph connected to a 5795C mass selective Triple‐Axis Detector (Agilent Technologies, United States). For that purpose, 1 μL of extract was injected at 250 °C in splitless mode on a ZB‐5MS column (Phenomenex, 30 m × 0.25 mm; ID 0.25 μm) with 5 m guard column with a constant flow of helium at 1 mL/min. The oven was programmed for 1 min at 45 °C and then subsequently ramped at 10 °C/min to 300 °C and kept for a final time of 5 min with a solvent delay of 5.5 min. The ionization potential was set at 70 eV, and scanning was performed from 45 to 400 atomic mass units, with a scanning speed of 3.99 scans/sec. Quantification was performed with an external calibration curve from α‐bisabolol (Sigma‐Aldrich). The Henry's law constants were calculated with the HENRYWIN program available online (https://www.epa.gov/tsca-screening-tools/epi-suitetm-estimation-program-interface ) at 25 °C [HENRYWIN v3.10] Bond Method.
6
In vivo Imaging of Olfactory Responses
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For live imaging of the olfactory sensory system, larvae were anesthetized (2% Tricaine Sigma) mounted in a cut tip of plastic Pasteur pipette in 2% low temperature agarose (Sigma) in embryo medium (Westerfield, 2007 ). The larvae were imaged in frontal view in an Attofluor Chamber (Thermo Fisher Scientific) filled with Embryo medium. The agarose covering the olfactory system was removed. Temperature was maintained at 26–28°C, and images were captured using a Spinning disc confocal microscope (Olympus) with a 20 × 0.95 NA water immersion LUMPlanFL/IR objective.
Time-lapse videos of copper exposure: To generate the time-lapse movies (Figures 5 , 6 , 8 , 9 ), stacks of images were collected with 3 μm/optical section in a total depth of 150-μm depth. All tracking data from time-lapse microscopy in control and copper-exposed larvae were processed using MTrackJ tracker in FIJI. Chemotactic index (CI) was calculated as described by Lämmermann et al. (2013 (link)), taking left or right OO as reference. Briefly, CI was defined as cos(α) with α as the angle between the distance vector to the damage site (OO) and the actual movement vector.
Time-lapse videos of copper exposure: To generate the time-lapse movies (
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