For DILS assays, seeds were surface sterilized and stratified at 4 °C for 3 d. Seedlings were then grown for 2 wk in large square Petri dishes containing 0.5× Murashige Skoog Basal Salts (Sigma #M6899), 0.5% (w/v) agar type E (Sigma #A4675), 0.05% (w/v) MES pH 5.7 (Sigma #M8250), at 16 h light/8 h dark, average lighting of 120 μmol m−2 s−1, 22 °C day/20 °C night, 55% humidity. Plates were covered with aluminium foil to create complete darkness for 3 or 6 d. For DILS in pots, 4-wk-old adult plants grown in jiffy pots were covered with a lid and completely wrapped in aluminium foil to create constitutive darkness.
Mes ph 5
Manufactured by Merck Group
Sourced in Germany
The MES pH 5.7 is a pH buffer solution produced by Merck Group. It is a laboratory reagent used to calibrate and maintain the pH of scientific instruments and experiments.
Automatically generated - may contain errors
2 protocols using mes ph 5
1
Standardized Seedling Growth for DILS Assays
2
Arabidopsis Calcium Influx Assay
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Calcium influx assays were performed as previously described in (Wanke et al. 2020). In brief, single 7-day-old Arabidopsis seedlings were placed into white 96-well plates filled with 200 µl reconstitution buffer (2.5 mM MES pH 5.7 [Sigma-Aldrich, Taufkirchen, Germany], 10 mM CaCl2 [Roth, Karlsruhe, Germany]). Before overnight incubation in the dark, the solution was replaced with 133 µl reconstitution buffer containing 10 mM coelenterazine (Roth, Karlsruhe, Germany). On the following day, chemiluminescence was measured using a TECAN SPARK 10M microplate plate reader. After the baseline measurement, 67 µl of three-fold concentrated elicitor solutions (or Milli-Q-water as a mock control) was manually added. Cytosolic calcium influx upon elicitor addition was constantly measured for 30 min. In order to assess undischarged aequorin for treatment normalization, 100 μl of 3 M CaCl2 (in 30% EtOH) was injected into each well, followed by constant measurement for 1 min. All steps were performed with an integration time of 450 msec.
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