The cells were removed from the filters as previously described (Sekar et al., 2004 (link)). Briefly, each quarter of a filter was further cut into 4 sections, transferred to a 2 ml reaction tube (Eppendorf) containing 1.5 ml of NaCl/Tween 80 (150 mM/0.05%) and incubated at 4°C for 20 min. The reaction tubes were attached to a rotating shaker with adhesive tape (Vortex Genie 2 with 3 inch platform, Scientific Industries; Time Tape, Milian) and vortexed at full speed for 15 min. The cell suspension was transferred to fresh reaction tubes and processed on the same day. If larger debris particles were present, an additional filtration step was introduced (Swinnex filter holders, diameter 13 mm; Whatman polycarbonate filters, pore size 0.8 μm). A detailed step-by-step protocol of the whole procedure is presented in Table 1 .
2 ml reaction tube
Manufactured by Eppendorf
Sourced in Germany
The 2 ml reaction tube is a laboratory consumable designed for various applications in scientific research and analysis. It provides a standard size container for holding and processing small liquid samples or reagents. The tube has a capacity of 2 milliliters and is made of high-quality, durable materials suitable for use in common laboratory equipment and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Tissuelyser 2, by Qiagen (2 mentions)
Polycarbonate filter, by Cytiva (1 mentions)
Axioskop 2 fs, by Zeiss (1 mentions)
Pico17, by Thermo Fisher Scientific (1 mentions)
1.5 ml reaction tube, by Eppendorf (1 mentions)
96 well microplate, by Bio-Rad (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
8 protocols using 2 ml reaction tube
1
Microbiome Cell Extraction from Filters
2
Direct Confrontation Co-Culture Assay
3
Evans Blue Dye Exclusion Assay for Cell Viability
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To determine mortality, the Evans Blue Dye Exclusion Assay was used with minor modifications according to our previous work6 (link): 2 ml of cell suspension were spun down in a 2-ml reaction tube (Eppendorf, Hamburg) at 4,000×g for 2 min (PICO 17, Thermo Scientific, Germany). Then, 1 ml of 2.5% Evans Blue (w/v, dissolved in double distilled water) was used to replace the supernatant. After staining for 5 min, Evans Blue was removed by centrifugation at 4,000×g for 1 min, and 1 ml of fresh basal medium was used to wash off the unbound dye. This washing step was repeated until the supernatant was colourless. Finally, the cells were concentrated in 200 μl of fresh basal medium, and 15 μl aliquots were scored for blue (dead) cells by means of a hematocytometer (Fuchs-Rosenthal) under an Axioskop microscope (Axioskop 2 FS; Zeiss, Germany) using a 20 × objective. Data represent mean values and standard errors of at least 1,500 cells per experiment and three independent experimental series.
4
Preparation and Characterization of Tattoo Pigments
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A tattoo matrix was simulated through mixing aqueous 20% solutions of glycerol and propylene glycol, ultrapure water and ACN as organic solvent in a volume ratio of 1 : 1 : 1 : 2. ACN was used in analogy to the preparation of the calibration mixture and MALDI matrices instead of typical tattoo solvents like EtOH or 2-propanol. Pure pigments were pre-dispersed in ultrapure water in a concentration of 100 mg mL -1 . Suspensions were further diluted with the self-mixed tattoo matrix to give final pigment concentrations of 80, 16 and 1.6 mg mL -1 , respectively.
For mechanical disruption, 1 mL pigment suspension was added to a 2 mL reaction tube (Eppendorf, Hamburg, Germany) together with 2 stainless steel beads (5 mm diameter, Quiagen, Hilden, Germany). Samples were homogenized at 30 Hz for 5 min in a TissueLyser II (Quiagen, Hilden, Germany).
For preparation of commercial tattoo inks, ca. 50-100 µl (1-2 drops) of ink is given into a 1.5 ml reaction tube (Eppendorf, Hamburg, Germany). To precipitate the pigments, 1 ml ultrapure water are added to the ink followed by centrifugation at 500g for 10 min. Before spotting, samples were washed by resuspending the pigment pellet in EtOH and centrifugation using the same parameters. The pellet was taken up in 0.5 mL EtOH.
For mechanical disruption, 1 mL pigment suspension was added to a 2 mL reaction tube (Eppendorf, Hamburg, Germany) together with 2 stainless steel beads (5 mm diameter, Quiagen, Hilden, Germany). Samples were homogenized at 30 Hz for 5 min in a TissueLyser II (Quiagen, Hilden, Germany).
For preparation of commercial tattoo inks, ca. 50-100 µl (1-2 drops) of ink is given into a 1.5 ml reaction tube (Eppendorf, Hamburg, Germany). To precipitate the pigments, 1 ml ultrapure water are added to the ink followed by centrifugation at 500g for 10 min. Before spotting, samples were washed by resuspending the pigment pellet in EtOH and centrifugation using the same parameters. The pellet was taken up in 0.5 mL EtOH.
5
Fluorescein Tyramide Amplification Assay
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The filters were cut into 4 equal pieces and transferred to 0.5 ml reaction tubes (Eppendorf) containing 500 μl of 20–100x diluted Anti-Fluorescein-HRP conjugate (Roche, Switzerland) in Tris/NaCl/blocking buffer (TNB, 100/150 mM/1%). Antibody binding was conducted over night at 4°C, followed by 3 washing steps in 1x PBS for a total of 20 min at 37°C. A secondary tyramide amplification step again using fluorescein labeled tyramides was conducted in 2 ml reaction tubes (Eppendorf) at 37°C in a water bath for 20 min, followed by 3 washing steps in 1x PBS for a total of 20 min.
6
DPPH Radical Scavenging Assay for Antioxidants
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The total free radical scavenging activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the method described by Brand-Williams et al. (1995). For each sample, 10 mg of lyophilized leaf powder were extracted in 1 mL 50% (v/v) methanol, shaken at 30 Hz for 2 min before centrifugation at 26,000× g for 10 min at 4 °C. The supernatant from each sample was transferred into new 2 mL reaction tubes (Eppendorf Hamburg, Germany). Incremental volumes of the supernatant up to 6 µL were pipetted into a 96-well microplate (Bio-Rad, Hercules, CA, USA), made up to 20 µL with 50% (v/v) methanol, and 200 µL DPPH solution was added (120 µM DPPH in 100% (v/v) methanol). The decrease in absorbance at 520 nm (i.e., radical scavenging) was followed for 30 min (Synergy HTX, Biotek, Winooski, VT, USA) until the linear decrease in absorbance ceased. Free radical scavenging activity for each sample volume was normalised to its protein content and calculated against a standard curve of ascorbate (0–8 nmols), and averaged.
7
Leaf Freezing and Grinding Protocol
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Leaves from untreated and from heat-hardened (H, H+D) individuals (n~25 for each treatment) were removed at midday, frozen in liquid nitrogen before freeze-drying for 3 days. Leaves from each replicate (n = 3–4, as indicated in figure legends) were ground to a fine powder in 2 mL reaction tubes (Eppendorf) with two 2 mm glass using a TissueLyser II (Qiagen, Düsseldorf, Germany) at 30 Hz for 6 min at freezing temperatures. Ground material was stored at −80 °C for 6–12 weeks before conducting measurements.
8
Reproducible Sampling of Freeze-Concentrated Liquid
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A hollow drill from Bürkle (Bad Bellingen, Germany) with an inner diameter of 8 mm has been used to take samples from the frozen bulk. A 3D-printed mount with nine drill holes in two different rows at an angle of 10.5° was placed on top of a chamber, providing reproducible drill positions across the whole chamber length. Under the assumption of negligible radial boundary effects, sampling from two different rows with overlapping sample volumes increased the sample resolution of a cross section as depicted in Figure 1 in solid and dashed lines. Samples were taken from the drill holes at three levels of 8 mm height with the last hole 2 mm above the chamber bottom. The sample above the first sample level has been discarded at all times to avoid uptake of ice fragments from the previous drilling and measuring of the freeze concentrated liquid from the center of the chamber that was pushed out where the expanding ice fronts met. The samples were transferred into 2 mL reaction tubes from Eppendorf (Hamburg, Germany) and thawed at room temperature. The buffer concentration of the samples was measured by analysis of the conductivity using a conductivity meter CDM 230 from Radiometer Analytical SAS (Lyon, France).
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