We tested several cell wall digestion enzymes from multiple manufacturers and found the performance of the enzymes varied between manufacturers, as well as between batches from the same manufacturers. We tested driselase (Sigma), cellulase (Sigma), cellulase R10 (Duchefa, Yakult), cellulase RS (Duchefa, Yakult), pectolyase (Duchefa; discontinued), pectinase (Sigma), macerozyme R10 (Duchefa) and hemicellulase (Sigma) for conventional whole-mount protocol. We chose cellulase RS (Duchefa), hemicellulase (Sigma) and pectinase (Sigma) or macerozyme R10 (Duchefa) for expansion microscopy based on their performance and availability.
Macerozyme r10
Manufactured by Duchefa Biochemie
Sourced in Netherlands
Macerozyme R10 is a mixture of cell wall degrading enzymes derived from the fungus Rhizopus. It is commonly used for the isolation and purification of plant protoplasts.
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Lab products found in correlation
Cellulase r 10, by Duchefa Biochemie (7 mentions)
Lsm 780, by Zeiss (2 mentions)
Yol1 34, by Bio-Rad (1 mentions)
Vectashieldtm, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Onozuka r10, by Duchefa Biochemie (1 mentions)
Miracloth, by Merck Group (1 mentions)
Cellulase onozuka rs, by Duchefa Biochemie (1 mentions)
Anti rat igg alexa fluor 488, by Cell Signaling Technology (1 mentions)
Cellulase onozuka r 10, by Duchefa Biochemie (1 mentions)
Trypsin, by Merck Group (1 mentions)
Cellulase, by Duchefa Biochemie (1 mentions)
Cellulase, by Merck Group (1 mentions)
Hemicellulase, by Merck Group (1 mentions)
Driselase, by Merck Group (1 mentions)
Pectinase, by Merck Group (1 mentions)
Cellulase rs, by Duchefa Biochemie (1 mentions)
Pectolyase, by Duchefa Biochemie (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Leptomycin b, by Cayman Chemical (1 mentions)
15 protocols using macerozyme r10
1
Optimizing Cell Wall Enzymatic Digestion
2
Nuclei Spread Preparation for Microscopic Analysis
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Rosette leaf material was harvested and directly fixed in ice-cold Carnoy’s fixative (3:1 ethanol/acetic acid) and kept at –20° until use. Nuclei spread preparations for microscopic analysis were made essentially as described by Schubert et al. (2001 (link)) and Tessadori et al. (2009 (link)) using modified enzymatic cell wall–degrading mixture [cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa) in 10 mM citrate buffer, pH 4.5] to digest cell walls. The air-dried slides were mounted in Vectashield (Vector Laboratories) with DAPI (2 µg ml-1) before observation and capturing.
For the (de)etiolation experiments, cotyledons of 5-d-old seedlings were fixed in 4% paraformaldehyde for 3 hr under white light condition or under a safe green light for the dark-grown seedlings, and treated with a solution containing 0.5% cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa), and 0.1% Triton X-100 for 1 hr. Cotyledons from at least three seedlings were isolated and squashed on a glass slide, flash frozen in liquid nitrogen, and incubated with PEMSB (50 mM Pipes, pH 7.3; 5 mM EGTA, pH 7.1; 5 mM MgSO4; 0.05% saponin; 5% wt/vol BSA) before being mounted with Vectashield (Vector Laboratories) supplemented with 2 μg/ml DAPI before observation and capturing.
For the (de)etiolation experiments, cotyledons of 5-d-old seedlings were fixed in 4% paraformaldehyde for 3 hr under white light condition or under a safe green light for the dark-grown seedlings, and treated with a solution containing 0.5% cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa), and 0.1% Triton X-100 for 1 hr. Cotyledons from at least three seedlings were isolated and squashed on a glass slide, flash frozen in liquid nitrogen, and incubated with PEMSB (50 mM Pipes, pH 7.3; 5 mM EGTA, pH 7.1; 5 mM MgSO4; 0.05% saponin; 5% wt/vol BSA) before being mounted with Vectashield (Vector Laboratories) supplemented with 2 μg/ml DAPI before observation and capturing.
3
Immunolocalization of α-Tubulin in Cuscuta
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Cuscuta root-like structure and shoot apex at 1st–7th day after germination were hand cut and fixed in 4% (w/v) paraformaldehyde in PEM buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, pH 6.8) for 1 h. After washing in PEM, cell walls were digested by a cocktail of 3% (w/v) macerozyme R10 and 3% (w/v) cellulase R10 (Duchefa) in PEM at room temperature for 1.5 h (modified according to Šamajová et al., 2014 (link)). The next steps were the incubation of the samples in absolute methanol (at -20°C) for 30 min and extraction with 5% (v/v) DMSO + 1% (v/v) Triton X-100 in PBS at room temperature for 1 h. Samples were subsequently incubated overnight with rat anti-α-tubulin antibody (YOL 1/34, Serotec) diluted 1:40 in PBS. Following PBS washing, the cells were incubated overnight with FITC-anti-rat antibody (Invitrogen) diluted 1:40 in the same buffer. DNA was counterstained with 250 μg/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma) in PBS for 10 min and after final washing the specimens were mounted in an antifade solution [0.5% (w/v) p-phenylenediamine in 70% (v/v) glycerol in PBS or 1M Tris-HCl, pH 8.0] or in the commercial antifade VECTASHIELDTM (Vector Laboratories).
4
Arabidopsis Mesophyll Protoplast Isolation and Transient Transformation
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Mesophyll protoplasts of Arabidopsis thaliana (Col-0) were isolated and transiently transformed according to Yoo et al. (2007) (link) and Costa et al. (2012) (link). Briefly, well-expanded rosette leaves from 3-to-5 week-old plants were cut into strips of 0.5–1 mm with a fresh razor blade. Leaf tissue was digested using an enzyme solution containing 1.25% cellulase Onozuka R-10 (Duchefa) and 0.3% Macerozyme R-10 (Duchefa) for 3 h at 23°C in the dark. The protoplast suspension was filtered through a 50 μm nylon mesh washed three times with W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7 adjusted with KOH) and used for PEG-mediated transformation. For each protoplast transformation 10 μg of a MidiPrep purified DNA (QIAGEN) plasmid harboring the 35S-CaMV::AtCRP1-GFP cassette was used. Protoplasts were maintained for 16–24 h at 23°C in the dark, before performing epifluorescent microscopy.
5
Isolation of Turnip Leaf Protoplasts
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Protoplasts from infected turnip leaves were obtained as described37 (link). Briefly, infected leaves were soaked in 2.5% diluted Domestos solution (http://www.unilever.com ) for 3 min and washed with water. Then the leaves were incubated with 1% cellulase R10 and 0.05% macerozyme R10 (http://www.duchefa-biochemie.com ) overnight, in the dark at 25 °C. The next day, protoplasts were filtered over one layer of Miracloth (http://www.merckmillipore.com ) and washed 3 times with protoplast medium by centrifugation at 80 g in a swing-out rotor for 5 min. Protoplasts were resuspended in protoplast medium and transferred to 2 ml Eppendorf reaction tubes. Before the experiments, protoplasts were incubated at room temperature with 5 rpm agitation for 1 h to allow recovery from the protoplast preparation procedure, as reported6 (link),7 (link).
6
Microfluidic Protoplast Isolation and Cultivation
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The digestion solution applied in the microfluidic experiments contained 4% cellulase Onozuka RS (Duchefa Biochemie, Haarlem, Netherlands) and 1% macerozyme R-10 (Duchefa Biochemie, Haarlem, Netherlands) in filter-sterilized F/2 medium, including 0.5 M sterile D-Sorbitol. The digestion solution was centrifuged at 10 000 g at 4 °C for 10 min in order to remove any potential impurities of the enzyme powder. The digestion solution was loaded into a syringe (Omnifix-F Solo Luer 1 mL, B. Braun), and the syringe containing the digestion solution was attached to the tubing of the microfluidic system and a constant flow rate was applied in order to move the enzyme solution through the microfluidic devices containing the trapped cells. After enzyme treatment, the digestion solution was replaced by washing solution and protoplasts were washed with continuous flow to remove the enzyme solution from the microfluidic devices. After 4-5 h of washing, regeneration medium was applied to the cells for 2 days, after which culture medium was added. All experiments were performed in triplicates.
7
Microtubule Visualization in Plant Cells
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For microtubule observations, control and extract-treated root rips were excised and fixed in 4% (w/v) paraformaldehyde (PFA) solution in PEM buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, pH 6.8) + 5% (v/v) dimethyl sulfoxide (DMSO) for 1 h. Fixed specimens underwent cell wall digestion with a 3% (w/v) Macerozyme R-10 + 3% (w/v) cellulase Onozuka R-10 (Duchefa Biochemie, Haarlem, The Netherlands) solution in PEM, for 90 min. After digestion, root tips were squashed gently on coverslips coated with poly-l -lysine and the released cells were left to dry and adhere. Afterwards, they were extracted with a 5% (v/v) DMSO + 1% (v/v) Triton X-100 solution in phosphate-buffered saline (PBS, pH 7.2), for 1 h. Rat anti-α-tubulin (YOL 1/34, Bio-Rad Laboratories, Hercules, CA, USA or Santa Cruz Biotechnology, Dallas, TX, USA) was used as primary antibody (diluted 1:50 in PBS, incubated overnight) and anti-rat IgG Alexa Fluor 488 (Cell Signaling Technology, Danvers, MA, USA) as secondary antibody (diluted 1:300 in PBS, incubated at 37 °C for 2 h). DNA counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, specimens were mounted with anti-fade medium (PBS 1:2 glycerol (v/v) + 0.5% (w/v) p-phenylenediamine).
8
Imaging of Cell Nuclei on Nanofiber Scaffolds
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Cell nuclei on nanofiber randomly oriented PVDF-TrFE scaffolds were imaged after staining for 10 min with DAPI (1 μg/ml), then placed in a 1.7-ml microcentrifuge tube with 1 ml of BY-2 medium containing either 0.05% trypsin (Sigma-Aldrich, T2601), 0.8% cellulase (Duchefa Biochemie, C8001), 0.2% Macerozyme R-10 (Duchefa Biochemie, M8002), or 1.5% pectate lyase (Megazyme, E-PLYCJ), and shaken at room temperature for 1 hour. Mock treatments used BY-2 medium alone. Scaffolds were washed in 25 ml of fresh medium three times and then stained for 10 min with DAPI for imaging.
9
Triticale Protoplast Transfection Protocol
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Methodology previously optimized for wheat protoplasts transfection [27 (link)] was adapted for triticale. Triticale protoplasts were transfected with preassembled vectors via polyethylene glycol (PEG)-mediated delivery. Seven days-old, etiolated seedlings of triticale cv. Bogo were used for protoplast isolation. The central part of the second leaves were chopped with new razor blades and incubated for 5 h in W5 containing cellulase R-10 (Duchefa) and macerozyme R-10 (Duchefa) at 25 °C. Protoplasts were then collected and immediately transfected with 25 µg of plasmid vector per 100 thousand cells. The four vectors were transfected using PEG treatment into triticale protoplasts. After 48 h incubation in darkness, at room temperature, without shaking, transfection efficiency was evaluated based on GREEN FLUORESCENT PROTEIN (GFP) expression. Genomic DNA was extracted from transfected protoplasts and used for PCR. Fragments of the ABA′OH-1 targeted sequences were amplified using PCR with the specific primers (Table S1 ), cleaned-up, re-annealed, and digested with T7 endonuclease. Digestion products were visualized with BioAnalyzer 2100 using High Sensitivity DNA chips. Cas9/gRNA activity was calculated based on the ratio of digested and undigested products.
10
BMS Protoplast Isolation and Swelling
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BMS protoplast isolation was performed as described previously (Moshelion et al., 2004 ). Briefly, protoplasts were isolated from 2 ml of 8‐d‐old BMS cell suspensions. The cells were left to sediment and the BMS medium was replaced with cell wall digestion solution (1 ml) (1.5% w/v cellulase Y‐C; Kyowa Chemical Products, Japan), 0.3% w/v Macerozyme R‐10 (Duchefa Biochemie, Haarlem, the Netherlands) in isotonic solution (to be described later). The cells were placed on a rotary shaker (90 rpm) for 2 h 30 min at 26°C. Then, the cells were passed through a nylon filter (20 μm pore size) and washed two times with isotonic solution. The protoplast swelling experiments were performed as described previously (Moshelion et al., 2004 ). The isotonic and hypotonic solutions (10 mM KCl, 1 mM CaCl2, 8 mM MES, pH 5.75) were adjusted with sorbitol to 300–330 and 150–160 mOsm, respectively. The Advanced Instruments 3300 Micro‐Osmometer (Advanced Instruments, MA, USA) served for all osmolarity measurements.
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