Quantification of Callose in Plant Tissues by Enzyme-Linked Immunosorbent Assay/ Immunofluorescence Spectrophotometry

Summary

This protocol quantifies callose in plant tissues using the callose-specific method, immunofluorescence spectrophotometry/ELISA, which is based on the binding of callose in the plant samples to callose-specific antibodies followed by quantification of bound callose through enzyme-labeled detection antibodies by measuring the resulting enzymatic activity.

Abstract

The existing methods of callose quantification from plant tissues include epifluorescence microscopy, fluorescence spectrophotometry, immunofluorescence microscopy, and indirect assessment of both callose synthase and β-(1,3)-glucanase activities. However, some of these methods have significant limitations, which include being time-consuming, non-specific to callose, labor-intensive, subjective, high autofluorescence, low sensitivity, being more qualitative rather than quantitative, and requiring the acquisition of software resources and technical skills. Therefore, there is a pressing need to explore alternative methods for callose quantification in plant tissues. It was hypothesized that immunofluorescence spectrophotometry or enzyme-linked immunosorbent assay (ELISA) that uses callose-specific antibodies could overcome some of the limitations of the current callose quantification methods. Biotic stress was administered by inoculating tissue culture-derived banana plantlets with Xanthomonas vasicola pv. musacearum (Xvm) bacteria which induced callose production. Banana corm tissue samples were collected at 14 days post-inoculation (dpi) for callose quantification using the new immunofluorescence spectrophotometry method. Callose production in the corms of Xvm-inoculated and control groups varied significantly in both the banana genotypes (independent sample t-test, p < 0.05). The immunofluorescence spectrophotometry method described here could be applied for the quantification of callose in different plant tissues with high specificity to callose, sensitivity, reliability, and reproducibility. Additionally, the use of a 96-well plate makes this method suitable for high throughput callose quantification studies with minimal sampling and analysis biases.

Introduction

Callose is a polysaccharide of β-1,3-glucan which is a naturally occurring compound found in the cell walls of a variety of higher plants. Callose is synthesized by callose synthases (CalS) and degraded by β-(1,3)-glucanases^1,2,3. It is involved in several biological processes, including growth and development and response to abiotic and biotic stresses^4,5,6. During pathogen infection, the increase in callose deposition in the papillae helps to prevent further microbial colonization, acting as a permeability barrier between the neighboring plant cells^{2,4,7,8,9,10,11,12}. By slowing down pathogen invasion in the attacked tissue, callose deposition allows time for the induction of additional defense responses¹³. Accurate spatiotemporal quantification of callose in the plant tissues is essential for understanding its role in the various physiological processes. However, the current methods of callose quantification from plant tissues, which include epifluorescence microscopy, immunofluorescence microscopy, and fluorescence spectrophotometry, possess significant limitations and quantification challenges. Therefore, there is a pressing need to explore alternative methods for callose quantification to overcome these drawbacks. It was hypothesized that immunofluorescence spectrophotometry or enzyme-linked immunosorbent assay (ELISA) could offer a more efficient method of callose quantification in plant samples, which includes high specificity, sensitivity, reliability, reproducibility and offers a high-throughput method.

The gold standard method for callose quantification involves imaging of aniline blue-stained callose particles by epifluorescence microscopy^14,15,16,17. The aniline blue-stained callose when exposed to blue or UV light excitation appears as yellow fluorescent particles, which can either be manually counted for the number of fluorescing callose particles^14,15 or counted by automated callose counting software. Manual counting of callose particles is laborious, time-consuming, and subjective to the researcher¹⁸, whereas automated counting requires acquiring software resources and a considerable technical skills. Some of the software used for automated callose counting include ImageJ^19,20,21,22, photoshop²³, CalloseMeasurer²⁴, Icy^18,25 and Ilastik²⁶. The aniline blue-stained callose may also be quantified using fluorescence spectrophotometry^{17,19,27,28,29}. However, the high background/autofluorescence associated with epifluorescence microscopy and fluorescence spectrophotometry make these methods difficult and unreliable due to low signal to noise ratio²⁶. Moreover, aniline blue can stain other β-1,3-glucans besides callose³⁰, making both epifluorescence microscopy and fluorescence spectrophotometry methods of callose detection and quantification partially non-specific and unreliable. A method of immunofluorescence microscopy for callose quantification which is based on callose-specific antibodies has also been used in several studies^31,32,33. Although this method shares a few disadvantages with epifluorescence microscopy of aniline blue-stained callose, it has the advantage of being callose-specific due to the antibodies used here. Callose quantification has also been performed using enzyme activity-based methods. Enzyme activity-based methods for callose quantification include assessment of callose synthase (CalS)^20,33 and β-(1,3)-glucanase activities^34,35. The enzyme activity-based methods have several disadvantages as compared to epifluorescence microscopy, immunofluorescence microscopy and fluorescence spectrophotometry. These include indirect estimation of callose, the difficulty of enzyme extraction and enzyme activity assays, and lack of even distribution of callose in the plant tissues.

Due to the disadvantages of the current methods of callose quantification which include epifluorescence microscopy, immunofluorescence microscopy, fluorescence spectrophotometry, CalS and β-(1,3)-glucanase assays, there was need to come up with a new method of callose quantification that would offer a number of advantages which include, 1) high specificity, sensitivity, reliability and reproducibility, and 2) easier callose quantification with high-throughput ability. The use of immunofluorescence spectrophotometry for callose quantification has not been explored. Here, a new method of callose quantification based on immunofluorescence spectrophotometry, more specifically, Enzyme-Linked Immunosorbent Assay (ELISA), is reported. The method can be optimized for the quantification of callose in different plants or their tissues with a good degree of specificity to callose, precision, reproducibility, and a high-throughput experimental capability. This method can also be modified to quantify any plant-based analyte if the antibody against that analyte is available. To ensure accurate, efficient, and reliable spatiotemporal distribution of callose in plant tissues, it is recommended that different methods of callose quantification are used simultaneously which include the microscopy, spectrophotometry and enzyme activity-based methods. Consequently, this will accurately and efficiently allow the understanding of the role of callose in various physiological and stress responses.

Protocol

1. Inoculation of experimental plants

1. Isolate, prepare and confirm the Xvm inoculum using standard procedures^36,37. Randomly select 10, 2-month-old tissue-culture-derived Musa balbisiana and Mbwazirume plants and inoculate the dorsal side of the youngest, fully open leaf petiole with 200 µL (1 x 10⁸ cells) of the PCR-confirmed Xvm inoculum (Figure 1)³⁸.
2. Similarly, randomly select 10 Musa balbisiana and Mbwazirume banana plantlets and inoculate them with 200 µL of sterile distilled water (SDW).
3. Maintain the Xvm-inoculated and control banana plants under similar conditions in the screenhouse with a 12 h light/12 h dark photoperiod at 25 °C, watering them every 3 days.
   ​NOTE: Refer to Table of Materials and Table 1 for the list of materials and equipment and recipe for buffers, respectively.

2. Sampling of corm tissues and sample preparation

1. Perform destructive sampling to obtain corm samples from the experimental plants (Figure 1).
2. Quickly excise approximately 2 g of the corm samples using sterile surgical blades at 14 days post-inoculation (dpi) and place them individually into labeled 50 mL tubes. Do this for all the 10 samples each collected from the Xvm-inoculated plants and the control plants.
3. Immediately immerse the 50 mL tubes containing the samples into liquid nitrogen.
4. Transport the samples to the laboratory and store them at -80 °C until ready to proceed. Store samples at −80 °C for up to 1 year or longer with minimal freeze-thaw cycles.
5. Freeze-dry the samples for 72 h using a freeze-drier (Figure 1).
6. Place exactly 30 mg of the freeze-dried samples into 2 mL tubes containing 2 steel bicycle beads and pulverize them into fine powder, with the speed set at 5 for 1 min (Figure 1).
7. Store the pulverized samples in a cool dry environment at room temperature, well-sealed until ready to proceed. Store the pulverized samples at room temperature for up to 4 months or at −80 °C for up to 1 year with minimal freeze-thaw cycles^39,40.

3. Extraction of callose and preparation of laminarin standards and blank

1. To extract callose from the 30 mg of the pulverized corm sample, add 800 µL of 1 M NaOH and incubate for 30 min at 80 °C in a water bath, with occasional vortexing after every 10 min^27,41 (Figure 1, Step 6).
2. Allow the samples to cool to room temperature (approximately 5 min) and then centrifuge at 15,294 x g for 5 min.
3. Transfer the supernatant (callose extract) to a sterile 2 mL tube and dilute with the blocking buffer (Table 1) at a ratio of 1:2.
   NOTE: Choose the dilution factor based on preliminary experimentation and optimization. Dilution of the callose extract may or may not be necessary. When needed, dilution should be performed in the blocking buffer and the dilution ratio optimized for different sample types to fall within the range of the standard curve.
4. Store the callose extracts at -20 °C until you are ready to proceed. Store the callose extract for up to 6 months with minimal freeze-thaw cycles.
5. Prepare a concentrated stock of laminarin standard at 100 mg/mL in 1 M NaOH^{6,42,43,44,45}.
   NOTE: Laminarin is a 1-3-β-glucan polymer and can be used as standard for callose equivalents in immunofluorescence spectrophotometry.
6. Incubate the resulting suspension of the standard at 80 °C in a water bath, gently shaking it at intervals, until all the laminarin is completely dissolved (approximately 20-40 min).
7. Cool the dissolved standard to room temperature (approximately 10 min).
8. Prepare laminarin standards at concentrations of 80 mg/mL, 60 mg/mL, 40 mg/mL, 20 mg/mL, 10 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.1 mg/mL, and 0.01 mg/mL using the blocking buffer, starting from the concentrated stock of 100 mg/mL.
9. Prepare the blanks by mixing 1 M NaOH and the blocking buffer at a ratio of 1:2. If dilution is required, use the same dilution ratio used to prepare the callose extract.
10. Store the laminarin standards and the blanks at −20 °C. Store standards and blanks for up to 6 months with minimal freeze-thaw cycles.

4. Callose quantification by the immunofluorescence spectrophotometry

1. Add 100 µL of the primary antibody (1-3-β-glucan-directed mouse IgG) prepared using the coating buffer (Table 1) to each of the 96 wells of the polystyrene ELISA plates (Figure 2).
2. Seal the plate tightly with paraffin film and incubate it overnight at 4 °C in a refrigerator.
3. The next day, place the plate on the bench and allow it to reach room temperature (approximately 10 min).
4. Wash the plate using standard blotting and washing procedures⁴⁶. Briefly, add 200 µL of the wash buffer (Table 1) to the wells, vortex it at 500 rpm for 30 s using a digital shaker and blot on tissue paper. Repeat this washing process 2x (Figure 2). Ensure the wells do not dry completely and use a sterile micropipette tip to remove any bubbles in the wells without touching the base and walls of the wells.
5. Add 200 µL of the blocking buffer to each of the wells (Figure 2) and seal the plate tightly with paraffin film. Incubate it for 4 h at 37 °C. Wash the plate as mentioned above (Figure 2).
6. Add 100 µL of the callose extract from the 10 Xvm-inoculated and 10 control plants to the designated wells. Add 100 µL of the laminarin standards to other wells. Add 100 µL of the blank to other wells (Figure 2).
7. Seal the plate tightly with paraffin film and incubate overnight at 4 °C in a refrigerator. Wash the plate again as mentioned above (Figure 2).
8. Add 100 µL of the primary antibody (1-3-β-glucan-directed mouse IgG) prepared using the blocking buffer to each of the wells (Figure 2).
9. Seal the plate tightly with paraffin film and incubate for 4 h at 37 °C, then wash the plate as indicated above (Figure 2).
10. Add 100 µL of anti-Mouse IgG-Alkaline phosphatase secondary antibody to each well^46,47 (Figure 2).
11. Seal the plate tightly with paraffin film and incubated overnight at 4 °C in a refrigerator. Then, wash the plate as indicated above (Figure 2).
12. Add 100 µL of freshly prepared para-nitrophenyl phosphate (pNPP) solution at a concentration of 1 mg/mL (Table 1) to the wells (Figure 2)
13. Incubate the plate on the bench at room temperature for 30 min⁴⁸.
    NOTE: The incubation time may vary depending on the plant samples and standard used. In this study, laminarin had the best reading at 30 min.
14. Terminate the reaction by adding 100 µL of freshly prepared stop solution (0.5 M NaOH) to each of the wells⁴⁸ (Figure 2).
15. Transfer the plate to a microplate reader (equilibrated to 37 °C), shake it at medium speed for 1 min, and read the absorbances at 405 nm⁴⁸ (Figure 1, Figure 2).

5. Data analysis

1. Perform a simple linear regression of laminarin absorbance at 405 nm against laminarin concentration (µg/mL) to obtain the standard curve. The equation of the standard curve is
   Y = β₁X + β₀
   where Y represents laminarin absorbance at 405 nm, β₁ is the slope of the regression line (Pearson correlation coefficient), X is Log₁₀ (laminarin concentration in µg/mL) and β₀ is the y-intercept.
2. Calculate callose concentrations in the corm samples by subtracting the absorbance of the blank from the absorbance of the samples. Then use the standard curve to estimate the callose concentration in µg/mL as laminarin equivalent (LE).
3. Examine all data for normality of distribution using the Shapiro-Wilk test and check for homoscedasticity of variances using the Levene's test (α > 0.05).
4. Utilize the independent sample t-test to compare the callose production between the Xvm-inoculated and control groups (p≤ 0.05).

Results

The known concentrations of the laminarin standards were analyzed through spectrophotometry and their corresponding absorbance values were recorded. Using this data, a Beer-Lambert plot was constructed by plotting the concentrations of the laminarin standards on the x-axis and the absorbance on the y-axis. The equation of the regression line was y = 0.5079x − 0.04534 (Figure 3)⁴⁹. The Beer-Lambert plot gave a high correlation coefficient (r) of 0.9988 and coefficient of determination (r²) of 0.9975 indicating a strong linear relationship between concentration and absorbance and up to 0.9975 of the variance in the dependent variable (absorbance) was explained by the independent variable (concentration; Figure 3; p<0.0001). Therefore, this model was a good fit, accurate and reliable for predicting the concentration of laminarin and callose. The concentration of callose in the banana corm samples were determined by measuring absorbance of the corm samples through spectrophotometry and using the absorbance values to calculate the callose concentrations using the equation.

The immunofluorescence spectrophotometry assay showed significant increase in induced callose in Xvm-inoculated plants as compared to the control group in both the banana genotypes (independent sample t-test, p < 0.05; Figure 4). Callose production in the control plants at 14 dpi was 1474.34 mg/mL and 1037.11 mg/mL for Mbwazirume and M. balbisiana, respectively (Figure 4). Inoculation of the plants with Xvm resulted in 4190.78 mg/mL and 1996.07 mg/mL of callose in the corms of Xvm-inoculated Mbwazirume and M. balbisiana plantlets at 14 dpi, respectively (Figure 4). Xvm inoculation caused a 1.9-fold to 2.8-fold higher callose production as compared to the control plants (independent sample t-test, p < 0.001; Figure 4).

Figure 1: Schematic illustration of the major steps involved in callose quantification by the new immunofluorescence spectrophotometry method. This figure demonstrates the major steps involved in callose quantification from banana tissues which include raising of the tissue culture derived plantlets, inoculation with Xvm, destructive sampling of the corm tissues, freeze-drying of the samples, extraction of callose and quantification of callose by the new immunofluorescence spectrophotometry method. This figure has been reprinted⁵⁰. Please click here to view a larger version of this figure.

Figure 2: Detailed schematic illustration of immunofluorescence spectrophotometry method for callose quantification. This figure gives a detailed demonstration of the immunofluorescence spectrophotometry method for callose quantification showing how the ELISA plate is loaded with the various components and how callose is finally quantified by spectrophotometry. This figure has been reprinted⁵⁰. Please click here to view a larger version of this figure.

Figure 3: Protein quantification. The Beer-Lambert plot based on laminarin standards of known concentrations and absorbance readings (at 405 nm) used for calculation of callose concentrations (as laminarin equivalents, LE) from absorbance readings of the corm samples. This figure has been reprinted⁴⁹. Please click here to view a larger version of this figure.

Figure 4: Comparison of callose concentration. Comparison of mean callose concentrations (mean ± SEM, n = 9) in the corms of M. balbisiana and Mbwazirume banana plantlets inoculated with Xvm as determined by the new immunofluorescence spectrophotometry method (independent sample t-test, α ≤ 0.05) Please click here to view a larger version of this figure.

Buffer	Composition			Storage
Blocking buffer	10 g of bovine serum albumin (BSA; 1% w/v), dissolved in 1 L of PBS.			-20 °C for up to 6 months
Coating buffer	1.59 g of sodium carbonate (Na2CO3), 2.93 g of sodium bicarbonate (NaHCO3), 0.2 g of sodium azide (NaN3), dissolve in 1 L of deionised H2O, pH 9.6.			-20 °C for up to 12 months
Conjugate buffer	2 g of PVP (2% w/v), dissolved in 1 L of blocking buffer.			-20 °C for up to 6 months
Para-nitrophenyl phosphate (pNPP) solution	Dissolve para-nitrophenyl phosphate (pNPP) in substrate buffer to a working concentration of 1 mg/mL			-20 °C for up to 12 months
Phosphate-buffered saline (PBS)	8.0 g of sodium chloride (NaCl), 0.2 g of monobasic potassium phosphate (KH2PO4), 1.15 g of dibasic sodium phosphate (Na2HPO4), 0.2 g of potassium chloride (KCl), 0.2 g of sodium azide (NaN3), dissolved in 1 L of deionised H2O, pH 7.4.			-20 °C for up to 12 months
Primary antibody in blocking buffer	Reconstitute the lyophilized primary antibody according to manufacturer’s instruction to obtain the concentrated stock. Dilute the obtained reconstituted primary antibody stock in blocking buffer to a working concentration of 2 μg/mL.			-20 °C for up to 6 months
Primary antibody in coating buffer	Reconstitute the lyophilized primary antibody according to manufacturer’s instruction to obtain the concentrated stock. Dilute the obtained reconstituted primary antibody stock in coating buffer to a working concentration of 2 μg/mL.			-20 °C for up to 6 months
Secondary antibody	1:1000 of secondary antibody concentrate: conjugate buffer			-20 °C for up to 6 months
Substrate buffer	97 mL of diethanolamine, 0.2 g of sodium azide (NaN3), dissolve in 1 L of deionised H2O, pH 9.8			-20 °C for up to 12 months
Wash buffer	0.5 mL of Tween 20 (0.05% v/v), dissolved in 1 L of PBS, pH 7.5			-20 °C for up to 12 months

Table 1: Recipe for buffers.

Discussion

The immunofluorescence spectrophotometry method was applied for the first time in quantification of callose in banana corms. It is based on enzyme immunoassay (EIA), used to quantify callose in the crude 1 M NaOH extract of banana using callose-specific antibodies^47,51,52. In brief, this new immunofluorescence spectrophotometry-based, callose-specific quantification assay involved coating of the microplate with callose-specific antibody which then binds to callose in the plant extract, followed by a secondary antibody also specific to callose. The detection of an antibody that is linked to an enzyme (alkaline phosphatase) is then added and forms a complex with the secondary antibody. A substrate, para-nitrophenyl phosphate (pNPP), is then added. The quantification of callose in the sample relies on the ability of alkaline phosphatase to cleave the phosphate groups from pNPP molecules, resulting in the formation of a yellow-colored compound (p-nitrophenol). The detection of the intensity of the yellow color was done using a spectrophotometer at 405 nm and the absorbance was then correlated to alkaline phosphatase activity, which was directly proportional to callose concentration present in the sample. The reaction was stopped by increasing the pH through addition of 0.5 M NaOH, which prevented further alkaline phosphatase activity.

In contrast, the standard-gold method for callose localization and quantification is the epifluorescence microscopy method, a well-established method based on staining callose with aniline blue fluorochrome⁵³. In brief, this method is based on the specific binding of aniline blue dye to beta-1,3-glucans (callose) forming a complex. The aniline blue in this complex is then excited using the ultraviolet light (wavelength of 365-375 nm). When the aniline blue dye is excited, it emits blue fluorescence that is captured to allow specific visualization of callose in plant tissues⁵³. Quantification of aniline blue-stained callose follows a series of imaging techniques that quantify the amount of fluorescence coming from the plant tissues. The fluorescence is proportional to the amount of callose available in the plant tissues. Epifluorescence microscopy has been used in visualization and quantification of callose in biotic stress⁵⁴.

In a separate study, callose deposition was determined using both epifluorescence microscopy and immunofluorescence spectrophotometry⁴⁹. While a few differences were observed in the data, statistical analysis largely showed congruence of epifluorescence microscopy and immunofluorescence spectrophotometry data, indicating consistency in the two methods of callose detection⁴⁹. Whereas results from the two methods were largely congruent, it is advisable that both epifluorescence microscopy and immunofluorescence spectrophotometry should complement each other for more reliable assessment. Even though the ELISA-based method is callose-specific, sensitive, and reliable, it is initially time-consuming during optimization of the protocol and getting a best-fit standard curve that fits all the concentrations and absorbances.

Some of the methods for callose quantification are laborious^17,32,55, time consuming^26,56, not callose-specific since aniline blue can stain other β-1,3-glucans besides callose³⁰, require expensive and sophisticated callose counting software^23,24,26, require considerate amount of technical skills and the high background/autofluorescence associated with fluorescence spectrophotometry makes callose quantification difficult and unreliable²⁶. Callose quantification methods based on automated image analysis workflow such as ImageJ software requires appropriate training of the image analysis plug-in such as the Trainable Weka Segmentation (TWS) for ImageJ and this is very cumbersome⁵⁷.

Immunofluorescence spectrophotometry-based assays are highly preferred due to their specificity, sensitivity, ease of use, and reproducibility. However, it is important to note that the detection system and its sensitivity may vary depending on the specific assay protocol and the equipment used.

Here, a new method of callose quantification based on immunofluorescence spectrophotometry is described. The method described here could be applied for the quantification of callose in different plant tissues and species with satisfactory level of specificity to callose, high precision and reproducibility. This method is an addition to the various callose quantification methods and can be used to monitor changes in callose levels during growth and development in plants and in response to various stimuli or treatments. Moreover, use of 96-well plate reduces subjective sampling and analysis making the method suitable for high throughput callose quantification. It is noteworthy that although callose may be easily extracted from freshly harvested plant tissues^17,27,55, the sample preparation step involving freeze-drying included as per this new protocol provides a leverage of keeping the samples for a very long time (up to 1 year) and allows storage and shipping of perishable biological samples between distant laboratories at ambient temperatures. This allows repeating callose extractions without the need for repeatedly setting up the entire experiment for fresh sample collection.

Immunofluorescence spectrophotometry is recommended for high throughput callose quantification experiments while epifluorescence microscopy and immunofluorescence microcopy can be used for both quantification of callose and histolocalization of callose in plant tissues. To ensure accurate, efficient, and reliable spatiotemporal distribution of callose in plant tissues, it is recommended that different methods of callose quantification are used simultaneously which include the microscopy, spectrophotometry and enzyme activity-based methods. Consequently, this will accurately and efficiently allow the understanding of the role of callose in various physiological and stress responses. Future research could comprehensively and simultaneously assess several methods for callose quantification which include epifluorescence microscopy, immunofluorescence microscopy, fluorescence spectrophotometry, immunofluorescence spectrophotometry and the enzymatic activity-based methods.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aluminium foil	Kim-Fay EA Limited, Nairobi, Kenya
Analytical balance	Mettler-Toledo AG, Greifensee, Switzerland	ML204/01
Beadbeater 96	BioSpec Products Inc., Bartlesville, OK, USA	1001EUR
Blotting paper	Kim-Fay EA Limited, Nairobi, Kenya
Bovine Serum Albumin (BSA)	Thermo Fisher Scientific, Massachusetts, USA	B14
Centrifuge, 5425R	Eppendorf, Hamburg, Germany	5406000240
Combined refrigerator- freezer (4 °C and −20 °C)	Haier Medical and laboratory Co. Ltd., Qingda, China	HYCD-282
Diethanolamine	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	191287
Digital Shaker, MixMate	Eppendorf, Hamburg, Germany	5353000529
Di-sodium hydrogen phosphate (Na2HPO4)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	141679
ELISA plates, 96-well, flat base, transparent, polystyrene, high binding	Sarstedt AG & Co. KG, Nümbrecht, Germany	82.1581.200
Filter for the iMark microplate reader, iMark 680, 405 nm	Lasec International Pty. Ltd., Cape Town, South Africa	BRD1681011
Freeze-drier, VirTis—BenchTop “K” series; Model: 4KBTXL	LabWrench, Canada, USA	448053
Hydrochloric acid (HCl)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	141020
Isotherm Forced Convection Incubator, IFA-54-8	Esco Lifesciences Group, Singapore	2100002
Laminarin	Alfa Aesar, Massachusetts, USA	J66193
Microcentrifuge tubes 1.5 mL	Eppendorf, Hamburg, Germany	022363204
Microcentrifuge tubes 2 mL	Genesee Scientific Corp., California, USA	24–283
Micropipette (30–300 μL), 12-channel, 2100 series	Eppendorf, Hamburg, Germany	EP-12-300R
Micropipette tips epT.I.P.S.  Singles	Eppendorf, Hamburg, Germany	022363204
Micropipettes (0.5–1000 μL), Research  plus	Eppendorf, Hamburg, Germany	3123000900
Microplate absorbance reader, iMark	Bio-Rad Laboratories, Inc., California, USA	1681135EDU
Microplate Manager 6 Version 6 software	Bio-Rad Laboratories, Inc., California, USA	1689520
Parafilm	Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany	740751
Para-nitrophenyl phosphate (pNPP)	Merck KGaA, Darmstadt, Germany	20-106
pH meter, HI9126	Hanna Instruments, Woonsocket, RI, USA	02310048991
Polyvinylpyrrolidone (PVP)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	A2260
Potassium chloride (KCl)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	A2939
Potassium di-hydrogen phosphate (KH2PO4)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	141509
Precision water bath, GP10	Thermo Fisher Scientific Inc., Newinton, CT, USA	TSGP10
Primary antibody (1-3-β-glucan-directed mouse IgG)	Bio supplies Australia Pty Ltd., Melbourne, Australia	400-2
Reagent reservoirs	Thermo Fisher Scientific Inc., Massachusetts, USA	15075
Secondary antibody conjugated to Alkaline Phosphatase (anti-Mouse IgG-Alkaline phosphatase)	Sigma Life Sciences, New Jersey, USA	A5153
Sodium azide (NaN3)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	A1430
Sodium carbonate (Na2CO3)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	141648
Sodium chloride (NaCl)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	A2942
Sodium hydrogen carbonate (NaHCO3)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	141638
Sodium hydroxide (NaOH)	PanReac AppliChem ITW Reagents, Química SLU, Barcelona, Spain	141687.1210
Tween-20	Biomatik Corporation, Ontario, Canada	A4031
Ultrapure distilled water	Thermo Fisher Scientific, Massachusetts, USA	10-977-015