A 6-mm RDEB skin biopsy was obtained with authorization from the National Research Ethics Services, Westminster (07/H0802/104), and with written informed consent from patients with RDEB-1 ((+/–) c.1732C>T p.R578X)/(+/–) c.2710+2T>C IVS20+2T.C) and RDEB-2 ((+/+) c.425A>G p.K142R). Excess connective tissue was removed using a sterile blade and the sample was incubated in neutral protease NB (1 unit/ml; SERVA Electrophoresis, Heidelberg, Germany) at 37 °C for 3 hours until the epidermis peeled off. The remaining dermis was fragmented and treated with collagenase NB6 (0.45 units/ml; SERVA Electrophoresis). The resulting cell suspension was seeded into a T25 flask and cultured at 37 °C in a 5% CO2 incubator.
Neutral protease nb
Manufactured by Serva Electrophoresis
Sourced in Germany
Neutral Protease NB is a lab equipment product that functions as a proteolytic enzyme. It is used to hydrolyze peptide bonds in proteins under neutral pH conditions.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase nb1, by Serva Electrophoresis (3 mentions)
Cmrl 1066 medium, by Corning (2 mentions)
Human serum albumin (hsa), by Baxter (2 mentions)
Collagenase nb6, by Serva Electrophoresis (1 mentions)
Collagenase nb4, by Serva Electrophoresis (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Celecoxib, by Selleck Chemicals (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
5 protocols using neutral protease nb
1
Isolation of Dermal Fibroblasts from RDEB Patients
2
Chondrocyte Isolation and Hypertrophy
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Primary chondrocytes were isolated from limb buds of E12.5 embryos by digestion with 1 U/ml Neutral Protease NB (Serva) for 15 min and with 0.3 U/ml collagenase NB 4 (Serva)/0.05% trypsin-EDTA (Life Technologies) for 30 min at 37°C. Single cell suspensions were obtained by passing through a 40-μm cell strainer. 2·107 cells were cultured in high-density as described previously [28 (link)]. For differentiation into hypertrophic chondrocytes, micromass cultures were cultured for 28 days. Afterwards, micromass cultures were fixed and stained with Alcian blue [28 (link)].
3
Peripheral Nerve Cell Isolation and Culture
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Fascicles remained in these culture conditions for 7–9 days, allowing for wallerian degeneration to take place.32 (link) After this period, all fascicles were collected into a 15-ml tube and centrifuged at 20g for 1 minute to estimate the packed fascicle volume (PFV). Seven milliliters of dissociation enzyme solution was then added to every 0.3 ml of PFV and placed in the incubator overnight at 37°C with 8% CO2. The dissociation enzyme solution was composed of 2 DMC U/ml of neutral protease NB (SERVA Electrophoresis GmbH) and 0.5 PZ U/ml of collagenase NB1 (SERVA Electrophoresis GmbH) in DMEM supplemented with 3.1 mM CaCl2 (International Medication Systems Ltd.). The next day, FBS was added to the fascicles to neutralize the enzymes.
The 15-ml tube was washed with DMEM containing 10% FBS (D10 medium) and then centrifuged at 150g for 5 minutes at 4°C to pellet the cells at passage 0 (P0). The P0 cells were then washed twice with D10 medium and resuspended in CM. The total viable cell number and viability were determined using methods described below in the Cell Viability and Counts section.
The 15-ml tube was washed with DMEM containing 10% FBS (D10 medium) and then centrifuged at 150g for 5 minutes at 4°C to pellet the cells at passage 0 (P0). The P0 cells were then washed twice with D10 medium and resuspended in CM. The total viable cell number and viability were determined using methods described below in the Cell Viability and Counts section.
4
Isolation and Characterization of Human Pancreatic Islets from Type 2 Diabetic and Non-Diabetic Donors
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Human pancreata were obtained between Dec. 2016 to Dec. 2018 from 17 T2DM and 12 ND organ donors with informed consent for research. Organ donor information was obtained and displayed in the Table 1 . The protocol of this study was approved by the Medical Ethics Committee of the Tianjin First Central Hospital (No.:2016N066KY). hND or hT2DM islets were isolated by Collagenase NB1 (SERVA, Heidelberg, Germany) and Neutral Protease NB (SERVA, Heidelberg, Germany) digestion followed by continuous density purification. High purity islets (>90%) were collected and cultured on CMRL-1066 medium (Corning, Manassas, VA, USA), supplemented with 10% Human Serum Albumin (Baxter, Vienna, Austria), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5% CO2.Table 1
Donor information.
| Donor Characteristics | Age (y) | BMI (kg/m2) | HbA1c (%) | Male (%) | Female (%) |
|---|---|---|---|---|---|
| 45.90±8.69 | 25.33±4.78 | 5.38±0.20 | 91.67% | 8.33% | |
| 52.53±9.27 | 25.34±2.50 | 7.76±1.45 | 76.47% | 23.53% | |
| 0.0783 | 0.9933 | 0.0002 |
5
Isolation and Stimulation of Human Islets
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Human islets were prepared by Collagenase NB1 (SERVA, Heidelberg, Germany) and Neutral Protease NB (SERVA, Heidelberg, Germany) digestion and continuous density purification. High purity islets (>80%) were collected and cultured in CMRL-1066 medium (Corning, Manassas, VA, USA), supplemented with 10% Human Serum Albumin (Baxter, Vienna, Austria), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in 5% CO 2 .
The human islets were incubated with 1 μM PGE2 (Sigma-Aldrich, Missouri, USA) or 10 ng/mL recombinant human IL-1β (Peprotech, New Jersey, USA) or a combination of 10 ng/mL recombinant human IL-1β and 10 μM Celecoxib (Selleck, Houston, Texas, USA) for 24 h.
The human islets were incubated with 1 μM PGE2 (Sigma-Aldrich, Missouri, USA) or 10 ng/mL recombinant human IL-1β (Peprotech, New Jersey, USA) or a combination of 10 ng/mL recombinant human IL-1β and 10 μM Celecoxib (Selleck, Houston, Texas, USA) for 24 h.
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