Brains and spinal cords were first finely minced using a sterile scalpel, washed with cold PBS, then processed based on the specific enzymes used. The commercially available Neural Tissue Dissociation Kit (P) (Kit) was used following the manufacturer's protocol (Neural Tissue Dissociation Kit (P); Miltenyi Biotec, San Diego, CA). One milliliter of accutase (Global Cell Solutions, Charlottesville, VA) was added to the tissues at 1 mL per tissue and incubated at room temperature for 15 (accutase 15), 30 (accutase 30), or 60 minutes (accutase 60). Twenty units of papain (Sigma-Aldrich) was added per tissue sample and incubated for 15 (papain 15), 30 (papain 30), or 60 minutes (papain 60) at 37°C. Immediately following the incubation, papain was quenched using 150 μg ovomucoid (Sigma-Aldrich). For the combination of accutase and papain (accutase 15/papain 30), 20 U of papain was first added to the brain isolates and incubated for 30 minutes at 37°C. After 30 minutes, the papain was quenched using 1.5 μg ovomucoid. The brains were then washed with cold PBS, and 1 mL of accutase was added for 15 minutes at room temperature. Following all enzymatic dissociation methods, brains were washed with cold PBS, and then subjected to 1 wash with 37% Percoll PLUS to remove remaining myelin. The myelin-free single cell suspensions were counted using a hemocytometer.
Papain
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Japan, China, Sao Tome and Principe, Ireland, Switzerland, Canada, Australia, Denmark
Papain is a proteolytic enzyme derived from the papaya fruit. It is a highly purified and concentrated form of the naturally occurring enzyme. Papain exhibits catalytic activity for the hydrolysis of peptide bonds in proteins.
Automatically generated - may contain errors
Lab products found in correlation
L cysteine, by Merck Group (41 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (39 mentions)
Bovine serum albumin (bsa), by Merck Group (38 mentions)
Trypsin, by Merck Group (38 mentions)
Pepsin, by Merck Group (37 mentions)
Neurobasal medium, by Thermo Fisher Scientific (35 mentions)
Glutamax, by Thermo Fisher Scientific (34 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (32 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (31 mentions)
Dnase 1, by Merck Group (24 mentions)
B27 supplement, by Thermo Fisher Scientific (20 mentions)
Neurobasal a, by Thermo Fisher Scientific (18 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (18 mentions)
Dmem f12, by Thermo Fisher Scientific (18 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (17 mentions)
Poly d lysine, by Merck Group (16 mentions)
Dnase, by Merck Group (16 mentions)
Poly l lysine (pll), by Merck Group (16 mentions)
Cysteine, by Merck Group (15 mentions)
Bromelain, by Merck Group (15 mentions)
754 protocols using papain
1
Enzymatic Dissociation of Nervous Tissue
2
Quantifying Sulfated Glycosaminoglycan Production
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On day 6, one half of each agarose construct was used to examine for PG content. Each sample was placed in 500 µL papain buffer (pH 7.4) containing 250 µg/ml papain (Sigma-Aldrich, St. Louis, Mo), 100 mM sodium phosphate, 10 mM EDTA and 10 mM cysteine HCl and incubated at 60 °C for 16 h. The amount of sulfated glycosaminoglycan in the digestion solution was determined by Dimethylmethylene (DMMB) blue dye-binding assay. Briefly, 20 µL aliquot of the papain digest was mixed with 200 µL of DMMB staining solution (pH = 1.5)85 . Absorbance of the mixture was read at 525 nm by the multimode plate reader. Chondroitin sulfate from bovine trachea (Sigma-Aldrich, St. Louis, Mo) dissolved in the same papain buffer was used as the standard. All measurements were normalized by DNA content as determined by the Hoechst 33258 fluorometric assay86 (link). The PG production was defined as an increased amount of PG content over 6 days by comparing with the PG content of agarose constructs on day 0.
3
Carbohydrate Extraction from Egg Shell Membrane
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Total protein was extracted from ESM by magnetic stirring 8 g ESM powder in 100 mL Tris-HCl buffer containing 4% SDS and 5.6 M urea for 4 days at 4°C. The mixture was centrifuged at 10,000× g for 30 min; then the supernatant was transferred to a new tube and further centrifuged for 30 min at 4000× g. The clear supernatant was dialyzed against dH2O using dialysis tubes (MwCO 12,000–14,000 Da; Sigma-Aldrich) for 3 days and then freeze-dried. To remove the protein cores, the lyophilized powder was incubated with 1 mg/mL papain (Sigma-Aldrich) in 0.05 M Tris-HCl pH 7.4 and 5 mM L-cystein (Sigma-Aldrich) at 37°C for 48 h, with 2× refreshing with papain during this period. The papain-degraded material was dialyzed (MwCO 6,000–8,000 Da; Sigma-Aldrich) against dH2O and freeze-dried. This fraction is called carbohydrate fraction and consists of various carbohydrate types, including glycans and GAGs >6,000 Da. The carbohydrate fraction was gamma sterilized before utilizing in cell experiments.
4
Papain-Mediated Fab Purification Protocol
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Papain (Sigma-Aldrich, cat.no. P4762) was dissolved in PBS with 20 mM L-cysteine to activate Papain. Antibody 28-12 was digested with Papain at an antibody-to-Papain ratio of 200:1 at 37 °C for 6 h. Protease activity was stopped by iodoacetamide (Sigma-Aldrich, cat. no. I6125). 28-12 was purified using protein A to remove the Fc region and undigested IgG antibody, followed by 2–3 rounds of dialysis using PBS at 4 °C for 6 h. Size-exclusion gel filtration chromatography was performed to further purify 28–12 Fab.
5
PG Sulfation Analysis of Cartilage
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For PG sulfation analysis, GAGs were extracted from cartilage and primary chondrocyte cultures. GAGs from femoral head cartilage of P4 mice were obtained by proteinase K digestion of the tissue as described above. Chondrocyte cultures were incubated with basal medium without FCS at 37 °C in 5% CO2 for 24 h. Then, the medium was made 0.1 M sodium acetate, pH 5.6, 5 mM EDTA and 5 mM cysteine and 20 U of papain (Sigma) were added, while the cell layer was scraped in papain digestion buffer (0.1 M sodium acetate, pH 5.6, 5 mM EDTA and 5 mM cysteine) and digested with 20 U of papain. Digestion was performed at 65 °C overnight.
papain and proteinase K in all digested samples (from cell cultures or from femoral head cartilage) were inactivated at 100 °C for 10 min and released GAGs were recovered and analysed by HPLC after 2-aminoacridone derivatization as previously described [43 (link)].
papain and proteinase K in all digested samples (from cell cultures or from femoral head cartilage) were inactivated at 100 °C for 10 min and released GAGs were recovered and analysed by HPLC after 2-aminoacridone derivatization as previously described [43 (link)].
6
Enzymatic Osteoarthritis Induction in Mice
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The KU Leuven Ethical Committee for Animal Research approved all animal experiments. Osteoarthritis was enzymatically induced in 8-week-old male wild-type C57/Bl6 mice by intra-articular injection of a mixture of 2% papain (5 µL) (Sigma) and its activator 0.03 M cysteine (5 µL) (Sigma) into the right knee joints on the first day as described before.31–33 (link) Control group received papain only, and the treated group received papain in combination with 0.1 mg suramin (Sigma). An equal volume of sterile PBS was injected into the left knee that served as a control knee. At day 3, mice received a second intra-articular injection of 0.1 mg suramin or PBS. Mice were sacrificed after 6 days, and hind limbs were isolated. These hind limbs were fixed with 4% paraformaldehyde (Millipore) overnight at 4°C. Whole knees were decalcified in 0.5 M EDTA (pH 7.5) for 15 days at 4°C and paraffin embedded to section at 5 µm in frontal plane for histology and TIMP3, VDIPEN and NITEGE immunohistochemistry. Samples were stained with Safranin O and haematoxylin for histology analysis. Articular cartilage damage was quantified by one blinded reader according to the OARSI (Osteoarthritis Research Society International) scoring system.34 (link) Two independent randomised experiments were performed and consisted a total of 30 mice.
7
Cartilage Extracellular Matrix Quantification
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The in vitro–formed cartilage and the tissue bridge were harvested separately and
each digested in 40 µg/mL papain (Sigma-Aldrich). The native cartilage removed
from the bone was digested in 80 µg/mL papain for 48 hours at 65°C as previously described.34 (link)
The DNA content of the papain digests was quantified using a fluorometric assay
(excitation, 356 nm; emission, 458 nm) and Hoechst 33258 dye (Polysciences) and
compared with a standard curve generated using serial dilutions of calf thymus
DNA (Sigma-Aldrich) as previously described.34 (link)
To quantify collagen content, papain digests were acid hydrolyzed for 18 hours at
110°C. Hydroxyproline content was measured using Chloramine-T/Ehrlich’s reagent
assay and spectrophotometry (λ = 560 nm) as previously described.34 (link)
A standard curve was generated with L-hydroxyproline (Sigma-Aldrich).
Sulfated glycosaminoglycan content in the papain digests was quantified using
dimethylmethylene blue dye and spectrophotometry (λ = 525 nm) and compared with
a standard curve generated using chondroitin sulfate (Sigma-Aldrich) as
previously described.34 (link)
each digested in 40 µg/mL papain (Sigma-Aldrich). The native cartilage removed
from the bone was digested in 80 µg/mL papain for 48 hours at 65°C as previously described.34 (link)
The DNA content of the papain digests was quantified using a fluorometric assay
(excitation, 356 nm; emission, 458 nm) and Hoechst 33258 dye (Polysciences) and
compared with a standard curve generated using serial dilutions of calf thymus
DNA (Sigma-Aldrich) as previously described.34 (link)
To quantify collagen content, papain digests were acid hydrolyzed for 18 hours at
110°C. Hydroxyproline content was measured using Chloramine-T/Ehrlich’s reagent
assay and spectrophotometry (λ = 560 nm) as previously described.34 (link)
A standard curve was generated with L-hydroxyproline (Sigma-Aldrich).
Sulfated glycosaminoglycan content in the papain digests was quantified using
dimethylmethylene blue dye and spectrophotometry (λ = 525 nm) and compared with
a standard curve generated using chondroitin sulfate (Sigma-Aldrich) as
previously described.34 (link)
8
Biochemical Analysis of Intervertebral Disc
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Tissue samples for biochemical analysis were obtained from consecutive 4 mm slices used for histological, immunohistochemical and FTIR imaging analyses. Tissue samples were harvested from the anterior AF, NP and posterior AF analogous to ROI 1, 3 and 5 respectively, as used for MRI T2* mapping. The samples were freeze dried (speed vac) and digested in 1.5 ml papain digestion solution containing 0.1 M sodium acetate, 0.01 M l -cysteine, 0.01 M EDTA and 0.33% papain (w/v) (all Merck Millipore, USA), the pH of the papain solution was titrated to 6.6 using 1 M NaOH. Samples were digested overnight at 65 °C in a continuously shaking warm water bath. GAG content was measured using a colorimetric 1,9 dimethyl-methylene blue assay according to the manufacturer's protocol (Biolcolor Ltd., Carrickfergus, UK). Hydroxyproline (HYP) content, representing total collagen content of the tissue, was quantified using a dimethylamino-benzaldehyde assay adapted from Paul et al.65 Both GAG and HYP were normalized by tissue dry weight (μg (mg DW)–1).
9
Quantification of GAG and DNA Content
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Pellets cultured for 1, 7 or 14 days were digested overnight at 60 °C in 100 μL papain digestion buffer (pH = 6.4) containing 0.1% papain, 10 mM EDTA-disodium salt (both from Merck), 100 mM sodium acetate, and 5 mM L-Cysteine·HCL (both from Sigma-Aldrich). Following digestion, samples were diluted 1:8 in water to measure the GAG content or samples were diluted 1:6 in Tris-EDTA (TE) buffer to measure DNA content. The GAG content was measured by adding 200 μL dimethylmethylene blue (DMB) solution (0.05 mM DMB, 41 mM NaCl, 45 mM glycin and pH = 3.0) to 40 μL papain-digested sample (pre-diluted 1:8 in water) and absorbance was measured at 590 nm using an iMark Reader (Bio-Rad). To determine the DNA content, PicoGreen® stock solution (ThermoFisher Scientific) was diluted 1:200 in TE buffer (10 mM Tris-HCl, 1 mM EDTA and pH = 7.5) and 50 μL of this solution was mixed with 50 μL papain-digested sample (pre-diluted 1:6 in TE buffer). After 5 minutes dark incubation at room temperature, fluorescence was measured at 485/520 nm (excitation/emission) with a CLARIOstar (BMG Labtech, Offenburg, Germany) using DNA obtained from human HEK293T cells to set the standard curve.
10
Biochemical Analysis of Intervertebral Disc Tissues
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Tissue samples for biochemical analysis were obtained from an adjacent slice to the histological section; anterior AF, NP, and posterior AF were harvested, that is, from MRI ROIs 1, 3, and 5, respectively. Samples were freeze-dried (speedvac) and subsequently digested in 1.5 mL papain-digestion buffer containing 0.1 M sodium acetate, 0.01 M L-cysteine, and 0.01 M EDTA, after which pH was titrated to 6.6 using 1 M NaOH and finally 0.33% (w/v) papain was added to the solution (all Merck Millipore). Samples were digested overnight in a continuously shaking water bath at 65 °C. papain digestion solutions were diluted and glycosaminoglycan (GAG) content was analyzed using a DMMB assay (Biocolor Ltd.) according to the manufacturer's protocol. The collagen content, expressed as the total amount of hydroxyproline (HYP), was quantified using a DMBA hydroxyproline assay, as described by Paul et al.35 (link) Measured amounts of GAG and HYP content were expressed in micrograms per milligram tissue dry weight.
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