All experiments using hAECs were performed with approval from the Monash Medical Centre Human Ethics Committee. Human AECs were isolated as previously described [13 (link)]. Briefly, placentae were obtained from women with uncomplicated pregnancies undergoing elective cesarean section at term. Women gave written, informed consent for the collection of their placenta. The amnion was manually stripped from the chorion, and the hAECs were enzymatically removed by two 1-h digestions using TrypZean (Sigma-Aldrich, St Lois, MO, USA). Following digestion, TrypZean was inactivated with Soybean trypsin inhibitor (Sigma-Aldrich) and the hAECs were collected by centrifugation (1800 g, 10 mins, RT). Live cell counts and viability were determined microscopically using trypan blue dye exclusion. Cells were cryopreserved using standard methods at 5 × 106 cells/ml. To thaw, hAEC sample tubes were quickly removed from liquid nitrogen and placed directly into a 37 °C water bath until thawed. Samples were washed with ice cold media to remove DMSO, and cell counts and viability were determined. Cells were then transplanted in experimental mice or analyzed in in vitro assays.
Trypzean
Manufactured by Merck Group
Sourced in United States, Germany
TrypZean is a laboratory equipment product manufactured by Merck Group. It is designed for the enzymatic digestion of proteins. The core function of TrypZean is to facilitate the breakdown of protein samples into smaller peptides, enabling further analysis and research.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Braunol, by B. Braun (2 mentions)
Braunoderm, by B. Braun (2 mentions)
Ultraculture media, by Lonza (1 mentions)
Rh bfgf, by Thermo Fisher Scientific (1 mentions)
Defined trypsin inhibitor, by Thermo Fisher Scientific (1 mentions)
Rhegf, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Proteasemax surfactant, by Promega (1 mentions)
Fetal calf serum (fcs), by Atlanta Biologicals (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
10 protocols using trypzean
1
Isolation and Characterization of Human Amniotic Epithelial Cells
2
Isolation and Culture of Human fRPCs
3
Protein Precipitation, Reduction, Alkylation, and Digestion
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HEK cell protein precipitates were resuspended by adding 200 µl of 8 m urea and 200 µl of 0.2% ProteaseMAX surfactant (Promega) dissolved in 50 mm NH4HCO3. The protein suspension was reduced by adding tris(2-carboxyethyl)phosphine (TCEP; Sigma-Aldrich) to 5 mm final concentration and incubated at 55°C with vigorous orbital shaking using a Thermomixer (Eppendorf). Protein alkylation was done by adding iodoacetamide (Sigma-Aldrich) to a 10 mm final concentration and incubating with vigorous rotation in the dark for 20 min. To digest the proteins, we added the following in order: 150 µl of 50 mm NH4HCO3, 2.5 µl of 1% ProteaseMAX dissolved in 50 mm NH4HCO3, and 1:100 (enzyme/protein, w/w) sequencing (seq) grade trypsin (Promega) to a final reaction volume of 500 µl. The digestion reactions were incubated for 3.5 h at 37°C with vigorous orbital shaking using a Thermomixer (Eppendorf).
For mouse brain cortex samples, TrypZean was used for protein digestion as previously published (McClatchy et al., 2020 (link)). Briefly, protein pellets were resuspended in an 8m urea, 100 mm Tris HCl, pH 8, buffer with sonication. Proteins were then reduced and alkylated as mentioned above and diluted in 100 mm Tris HCl buffer to dilute urea to 2 m . TrypZean (Sigma-Aldrich) was added 1:25 (enzyme/protein, w/w), and samples were incubated at 37°C overnight with vigorous shaking.
For mouse brain cortex samples, TrypZean was used for protein digestion as previously published (McClatchy et al., 2020 (link)). Briefly, protein pellets were resuspended in an 8
4
PR8OVA323 Influenza Virus Generation
5
Autologous Bone Marrow-Derived Mesenchymal Stem Cell Transplantation
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Bone marrow (30 ml) was collected from the humeral head of the pigs 12 days before injection, as previously described 16 . The four autologous BM‐MSC transplantations required two humeral bone marrow collections (BM1 and BM2). Autologous bone marrow mononuclear cells (BM‐MNCs) were expanded in a clinical‐grade BM‐MSC production process that used 636 cm2 closed culture devices (Cellstacks, Macopharma, Tourcoing, France) and were seeded at 100,000 cells/cm2 in GMP minimal essential medium α (α‐MEM, Macopharma) supplemented with 10 µg/ml ciprofloxacin (200 mg/100 ml, Aguettant, Lyon, France) and 20% fetal bovine serum (A15–101 PAA, France). After 3–4 days, nonadherent cells were removed and cultures were refed with fresh medium. Thereafter, cultures were fed twice a week, and the adherent fibroblast‐like cells (passage 0) were cultured for 2–3 weeks. At that time, they were detached by using recombinant bovine trypsin expressed in corn (Trypzean, Sigma‐Aldrich, USA) and numerated by Trypan blue staining. Cells were suspended in a saline solution (0.9% NaCl) supplemented with 0.4% final human albumine (LFB, France) prior to animal administration.
6
Skin Biopsy and Cell Isolation
7
Enzyme Screening for Cell Detachment
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Five different enzymes were tested for detachment potential, Trypsin 0.25% (Gibco Cat. #25200) and Trypsin 0.05% (Gibco Cat. #25300), TrypZean (Sigma Cat. #T3449), TrypLE (Gibco Cat. #12605), and Accutase (Invitrogen Cat. #00–4555-56). Samples were taken from the bioreactors and harvested in conical tubes. For the enzyme screening experiments, an exposure time of 9 min was used, then for the following experiment analyzing exposure times, time points of 3, 6, 9, 12, and 15 min were used. The cell suspension was then filtered through a 70 μm sieve (Falcon Cat.# 352,350) and cells were enumerated on a haemocytometer using 0.1% trypan blue exclusion. The harvesting efficiencies were calculated by dividing the number of cells recovered to the attached cell density number that was obtained using the crystal violet nuclei method.
8
Isolation and Culture of Human Retinal Progenitor Cells
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All human material work was performed with the approval of the Institutional Review Board of Harvard Medical School. hRPCs were isolated from human fetal neural retina at 16 weeks’ gestation as previously described [9 (link)]. Cells were cultured onto fibronectin (Akron)-coated flasks (surface 75 cm2, vented cap, sterile, Nunclon Delta) in Ultraculture medium (Lonza), supplemented with 10 ng/mL recombinant human basic FGF (Peprotech), 2 mM L-glutamine (Invitrogen), 20 ng/mL recombinant human EGF (Peprotech), and 0.4 mM Primocin (Invitrogen) in a low-oxygen incubator (37 °C, 5% O2 5% CO2, 100% humidity) as a monolayer culture to achieve high density. Upon reaching 80% confluence, cells were passaged using 10X TrypZean (Sigma-Aldrich, St. Louis, MO, USA) and HBSS (Hank’s Balanced Salt Solution, no calcium, no magnesium, ThermoFisher, Waltham, MA, USA). Cell number and viability were estimated at each passage, using Trypan blue (Sigma-Aldrich) and a hemocytometer (Countess™ II FL Automated Cell Counter, Thermo Fischer Scientific). Cells were then re-plated onto a fibronectin coated T75 surface at a density of 15,000 cells/cm2 in the same medium. All work was performed with expanded hRPCs at passage 10.
9
Tissue Proteome Profiling by TMT Labeling
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Liver samples were lysed 10 min at 95 C in 5% SDS, 50 mM TEAB pH 7.55. After cooling, DNA was sheared by 10 min of sonication. Protein concentration was determined using micro BCA using BSA as standard. Fifty µg of each sample were digested by means of the Protifi™ S-Trap™ Mini Spin Column Digestion Protocol. Briefly, proteins were reduced and alkylated (15 mM TCEP, 25 mM CAA) 1 h at 45 C in the dark. SDS was removed from samples in the S-Trap column using 90% methanol in 100 mM TEAB and proteins were digested with 125 µl of trypsin in 50 mM TEAB pH 7.55 (Trypzean, Sigma, protein:enzyme ratio 1:25, 16 h at 37 C). The resulting peptides were eluted from S-Trap columns, speed-vac dried, and re-dissolved in 20 µl of 100 mM HEPES pH 8.0. 50 µg per sample were labelled using Thermo Scientific TMT11plex™ Isobaric Label Reagent Set following manufacturer’s instructions. Samples were mixed in 1:1 ratio based on total peptide amount, which was determined from an aliquot by comparing overall signal intensities on a regular LC–MS/MS run. The final mixture was finally desalted using a Sep-Pak C18 cartridge (Waters) and dried prior high pH reverse phase HPLC pre-fractionation.
10
Skin Biopsy and Cell Isolation
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Skin sampling occurred in accordance with the German Act on Organ and Tissue Donation, Removal and Transplantation (Transplantationsge-setz). After obtaining written informed donor consent, an elliptical ~3 × 1.5 cm2 excisional skin biopsy was taken from behind the ear. Donors who were serologically positive for HIV1/2 or had active hepatitis B virus, hepatitis C virus or active syphilis (Clinical Laboratory Improvement Amendments-positive/Treponema pallidum antibodies ≥1:80/fluorescent treponemal antibody IgM/rapid plasma reagin) were excluded. The manufacturing process took place in a European Union GMP grade B clean room facility under laminar air flow (A in B). Biopsy tissue was disinfected using povidone-iodine solution (Braunol and Braunoderm; B. Braun, Melsungen, Germany), washed, dissected and subjected to two-step enzymatic digestion using collagenase (Collagenase NB 6 GMP Grade; Nordmark, Uetersen, Germany), followed by non-animal recombinant trypsin (TrypZean; Sigma-Aldrich, Taufkirchen, Germany). Following filtration and washing/centrifugation, pellets were resuspended in a stem cell-favoring medium.
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