An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation

Protocol

1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation

1. Plant growth and maintenance.
   1. To synchronize germination, stratify Arabidopsis seeds by suspending approximately 50 seeds in 1 mL of sterile 0.1% agar [w/v] and store at 4 °C (no light) for 3-4 days.
      NOTE: Stratify a wild-type background control (for example, Col-0) and genotypes with high and low immune outputs (for example, cpk28-1 and bak1-5, respectively) to serve as internal controls.
   2. Sow seeds on the soil and germinate under standard short-day conditions (22 °C,....

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Results

Figure 1. Luminol-based oxidative burst assay following immune induction in Arabidopsis. (A) Grow plants on soil in short-day conditions for 4-5 weeks. (B) Use a 4 mm biopsy punch to collect leaf discs from each plant and recover overnight in ddH2O. (C) Add reaction solution (100 μM luminol, 10 μg/mL HRP, and the desired concentration of elicitor) and measure light emission over 40-60 min, in 2 min intervals with an integration time of 1,000 ms. (D) Determine average photon counts for each ....

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Materials

Name	Company	Catalog Number	Comments
20-20-20 Fertilizer	Plant Prod	10529	Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.
4 mm Biopsy Punch	Medical Mart	232-33-34-P	A cork borer set with a 0.125 cm^2 surface area can also be used.
BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL)	Sartorius	725240	Any multichannel pipette can be used, as can a single pipetter if necessary.
elf18 (Ac-SKEKFERTKPHVNVGTIG)	EZ Biolab	cp7211	Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.
Forceps	Fisher Scientific	22-327379
Horseradish Peroxidase	Sigma-Aldrich	P6782	Dissolve in pure water. Store at -20C and away from light.
Luminol	Sigma-Aldrich	A8511	Dissolve in DMSO. Store at -20C and away from light.
Murisage and Skoog Basal Salts	Cedarlane Labs	MSP09-100LT	Store at 4C.
Soil	SunGrow Horticulture	Sunshine Mix #1	Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.
SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module	Molecular Devices		Any plate reader capable of detecting luminescence can be used for these assays.
White Polystyrene 96-Well Plates	Fisher Scientific	07-200-589