Chromosome spreads were prepared according to Waminal et al. (2012 (link)) and Eliazar et al. (2019 (link)). Briefly, the meristematic tips were immersed in an enzyme solution consisting of 1 % pectolyase Y-23 (Duchefa Biochemie, Netherlands) and 2 % cellulase R-10 (Duchefa Biochemie, Netherlands) for 60–90 min at 37 °C. Next, chilled Carnoy’s solution was added, and after centrifugation, the precipitate was pipetted and dropped onto pre-warmed glass slides in a humid chamber at 70 °C and then air-dried.
Cellulase r 10
Manufactured by Duchefa Biochemie
Sourced in Netherlands
Cellulase R-10 is an enzyme preparation derived from the fungus Trichoderma reesei. It is formulated to hydrolyze cellulose into glucose and other soluble carbohydrates.
Automatically generated - may contain errors
Lab products found in correlation
Macerozyme r10, by Duchefa Biochemie (7 mentions)
Pectolyase, by Merck Group (5 mentions)
Cytohelicase, by Merck Group (5 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Pectolyase y 23, by Duchefa Biochemie (1 mentions)
Yol1 34, by Bio-Rad (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Miracloth, by Merck Group (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)
13 protocols using cellulase r 10
1
Chromosome Spread Preparation Protocol
2
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).
3
Immunolabeling of Grass CENH3 Proteins
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Root tips pretreated using nitrous oxide at a pressure of 10 bar for 2 h were fixed in 4% (w/v) paraformaldehyde (PFA) prepared in 1× phosphate-buffered saline (PBS) for 20 min on ice. Next, roots were weakly vacuumed and digested in an enzyme solution (1% pectolyase (w/v) (Sigma), 1% (w/v) cytohelicase (Sigma), 0.7% (w/v) cellulase R-10 (Duchefa), and 0.7% (w/v) cellulase (Calbiochem)) in 1× PBS for 2 h. The roots were then washed in 1× PBS on ice for 5 min twice and squashed on slides in 1× PBS + 0.001% (v/v) Tween 20 using a coverslip. Cover slips were removed after freezing in liquid nitrogen, and slides were immediately stored in 1× PBS. Immunolabeling was performed according to Manzanero et al. (2000 (link)), except that the slides were treated using a microwave 800 W for 1 min in 10 mM sodium citrate buffer to improve the chromatin accessibility (Ruban et al. 2020 (link)) and finally kept in 1× PBS for 5 min. Rabbit anti-grass CENH3 (Sanei et al. 2011 (link)) (diluted 1:2000) and donkey anti-rabbit Alexa Fluor 488 (Jackson ImmunoResearch) (diluted 1:100) were applied as primary and secondary antibodies, respectively. To distinguish between A- and B-located CENH3 signals, after immunostaining FISH using the B-specific probe, Fp-Sat253 was performed as described by Ishii et al. (2015 (link)).
4
Metaphase Arrest in Nigella Roots
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Nigella seeds were germinated on moist filter paper in petri dishes for 3–6 days at room temperature. Roots were subjected to nitrous oxide (N2O) gas at 10 bar pressure for 2 h to arrest dividing cells at metaphase. Treated roots were fixed in ice-cold 90% acetic acid for 10 min, then transferred to 75% ethanol and stored at −20°C until use. Roots were first washed in ice-cold water, followed by 0.01 M citrate buffer (0.01 M citric acid and 0.01 M sodium citrate, pH 4.8) each for 10 minutes. Root meristems were placed in a microtube containing 30 μl enzyme mixture [0.7% cellulase (CalBiochem 219466), 0.7% cellulase R10 (Duchefa C8001), 1% cytohelicase (Sigma C8274) and 1% pectolyase (Sigma P3026) in 0.01 M citrate buffer] and were digested at 37°C for 60 to 90 minutes. Slides were prepared using the dropping method according to Abdolmalaki et al. (2019) (link). The specimens were fixed in 4% paraformaldehyde in 1 × PBS (3 mM NaH2PO4, 7 mM Na2HPO4, 0.13 M NaCl, pH 7.4) for 10 min at room temperature, followed by washing in 2 × SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0) and dehydrating in 96% ethanol.
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
Flow-Sorted Chromosome Embedding Protocol
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Selected roots were washed twice in ice water for 5 min and once in a citric buffer for 5 min. The roots were digested in an enzyme mixture [1% pectolyase (Sigma); 1% cytohelicase (Sigma); 0.7% cellulase R-10 (Duchefa) and 0.7% cellulase (Calbiochem) in 0.1 M citric buffer] at 37°C for 45 min. Following enzymatic digestion, the roots were washed twice in ice-cold water for 5 min and twice in ice-cold ethanol for 5 min. Root tips without root caps were collected into the tube containing ethanol:acetic acid (1:3) and mashed. 7 μl of this suspension was dropped onto a cold wet slide, transferred to a hot plate (55°C) and fixed with 25 μl of ethanol:acetic acid (3:1). The slides were allowed to dry at least for 1 h at RT.
To preserve the native volume of the chromosomes, metaphase chromosomes were flow-sorted into 1× meiocyte buffer A (1× buffer A salts, 0.32 M sorbitol, 1× DTT, 1× polyamines) and subsequently embedded into a 5% polyacrylamide gel as described (44 (link),45 ) with minor modifications (46 (link)).
To preserve the native volume of the chromosomes, metaphase chromosomes were flow-sorted into 1× meiocyte buffer A (1× buffer A salts, 0.32 M sorbitol, 1× DTT, 1× polyamines) and subsequently embedded into a 5% polyacrylamide gel as described (44 (link),45 ) with minor modifications (46 (link)).
7
EdU-Based DNA Replication Analysis
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DNA replication analysis was performed with the EdU kit (BCK-EdU 594-1, baseclick GmbH, Germany) following the manufacturer’s protocol. Root tips were collected and incubated in a humid chamber with a filter paper embedded in an EdU solution for 3 h at room temperature for the incorporation of the dNTP analog. After incorporation, root tips recovered in water for 30 min, were pretreated in 8-hydroxyquinoline for 24 h at 10°C, and were fixed in ethanol:acetic acid (3:1 v/v). The cell walls were digested by treating the root tips with an enzyme mix of 0.7% cellulase R10 (Duchefa C8001), 1% pectolyase (Sigma P3026), and 1% cytohelicase (Sigma C8274) for 90 min at 37°C. The squashing of chromosomes was performed in a drop of 45% acetic acid. Then, the slides were immersed into liquid nitrogen to remove the coverslips. The slides were incubated with 3% BSA for 20 min at room temperature and then with the detection mixture for 30 min at room temperature in the dark. Sequential FISH with the Ebusat 1 and Ty3/Gypsy-Tat LTR-RT probes was performed as described above. Images were captured as described above.
8
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.
9
Optimizing Cell Wall Enzymatic Digestion
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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.
10
Optimized Protoplast Isolation Protocol
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Fresh enzyme solutions were prepared before the isolation of the protoplasts. To standardize the protoplast isolation for selected tea cultivars, three enzymes 1–3% cellulase R-10 (Duchefa Biochemie), 1% hemicellulase and 0.2-1% macerozyme R-10 (Duchefa Biochemie) were used. The enzymes were dissolved in a protoplast salt solution (modified from Xu et al., 2021) containing 20 mM 2-ethanesulfonic acid (pH 5.7), 0.6 M mannitol, 10 mM CaCl2, 20 mM KCl and 0.1% bovine serum albumin. The 4% polyvinylpyrrolidone (PVP) was also added to prevent the oxidation of the phenols, as oxidized phenol may interfere with protoplast isolation. The different mannitol concentrations were tested in the range of 0.2-1.0 M for optimum osmotic pressure to enhance the integrity of the isolated protoplast. The solution containing the enzymes was heated at 55ºC for 15 min to dissolve and activate the enzymes. The enzyme solution was cooled down to room temperature and then filtered through 0.22µ syringe filters. The filter-sterilized enzyme solution was further used for the hydrolysis of leaf tissue.
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