Our protocol for isolation of mouse primary pancreatic acinar cells has been previously described in detail.18 (link) For 3D culture, acinar cells were seeded in a mixture of rat tail collagen I/Waymouth media without supplements on cell culture plates coated with rat tail collagen I (BD Biosciences, San Jose, Calif). TGF-α (R&D Systems, Minneapolis, Minn) or TGF-β1 (Preprotech, Rocky Hill, NJ) were added to the media on top at 50 ng/ml. At the endpoint viability of cells was confirmed using Hoechst 33342 (Invitrogen, Carlsbad, Calif). All samples/experimental conditions were performed in triplicates and, after five days of 3D culture, numbers of ducts per field (whole well) for each condition were determined and photos were taken to document cellular structures. The experiment was performed in three replicates using pancreata of different individual mice.
Rat tail collagen 1
Manufactured by BD
Sourced in United States, Canada, United Kingdom, Belgium, France, Uruguay
Rat tail collagen I is a naturally-derived extracellular matrix protein isolated from the tails of rats. It is a fibrous protein that provides structural support and promotes cell adhesion. The core function of rat tail collagen I is to serve as a versatile biomaterial for various cell culture and tissue engineering applications.
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Lab products found in correlation
Matrigel, by BD (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Soybean trypsin inhibitor, by Thermo Fisher Scientific (3 mentions)
Tgf β1, by Thermo Fisher Scientific (2 mentions)
Female swiss mice, by Janvier Labs (2 mentions)
Hepa1 6 cells, by American Type Culture Collection (2 mentions)
Phalloidin tritc, by Merck Group (2 mentions)
Fibronectin, by BD (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Ethanol, by Thermo Fisher Scientific (2 mentions)
Sigmacote, by Merck Group (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Slygard 184, by Thermo Fisher Scientific (2 mentions)
Pluronic f108 nf, by BASF (2 mentions)
107 protocols using rat tail collagen 1
1
Isolation and 3D Culture of Mouse Pancreatic Acinar Cells
2
Autologous Muscle Graft Transplantation for VML Repair
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Autologous minced grafts were derived from the original TA muscle tissue that was excised to make the VML defect. The excised muscle tissue was minced into ∼1 mm3 pieces. The minced grafts were then suspended in a collagen hydrogel; rat tail collagen I (Becton Dickinson) was diluted in 2.5× Dulbecco's modified Eagle's medium (Gibco) to create a 3 mg/mL solution and kept on ice while the minced graft was prepared. After mincing, the desired volume (0–50%) of muscle tissue was added to a well of a 48-well culture plate. A volume of collagen hydrogel was added to the well to bring the final volume to 400 μL (i.e., the volume of muscle tissue was calculated based on muscle density), which filled the defect area. The mixture was stirred to create a homogenous distribution of minced grafts within the construct. The collagen constructs were then allowed to crosslink at 37°C for ∼45 min, after which time the constructs were transplanted to the VML defect. Fascia and skin were closed by suturing and stapling each layer, respectively.
3
Isolation of Murine Epidermal Keratinocytes
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Isolation of primary epidermal keratinocytes was performed as described earlier (23 (link)). Briefly, disinfected tail skins of adult mice were digested overnight at 4˚C with trypsin (0.25%; w/o CaCl2, Gibco, Thermo Fisher Scientific, USA). Detached epidermal cells were seeded overnight at 32°C, 5% CO2 in minimum essential medium with Earle`s Balanced Salt Solution medium (Lonza, Switzerland) containing 0.2 mM CaCl2 (Merck, Germany). Subsequently 1x106 isolated cells were cultured on fibronectin (Roche, Switzerland)/rat tail collagen I (Becton Dickinson, Corning, USA)-coated six well-culture plates in Keratinocyte Growth Medium 2 with supplement mix (PromoCell, Germany) to the confluence of 70–80%. Culture and stimulation conditions in Keratinocyte Growth Medium 2 (PromoCell, Germany) are indicated in Supplementary Figure 1
4
Propagation and Cultivation of Rodent Malaria Parasites
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P. berghei and P. yoelii blood stage parasites were propagated in female Swiss mice (6–8 weeks old, from Janvier Labs). We used wild type P. berghei (ANKA strain, clone 15cy1) and P. yoelii (17XNL strain, clone 1.1), and GFP-expressing PyGFP and PbGFP parasite lines, obtained after integration of a GFP expression cassette at the dispensable p230p locus.25 (link)
Anopheles stephensi mosquitoes were fed on P. berghei or P. yoelii-infected mice using standard methods,72 (link) and kept at 21°C and 24°C, respectively. P. berghei and P. yoelii sporozoites were collected from the salivary glands of infected mosquitoes 21–28 or 14–18 days post-feeding, respectively. P. berghei and P. yoelii sporozoite infections were performed in female C57BL/6 or BALB/c mice, respectively (6 weeks old, from Janvier Labs), by intravenous injection in a tail vein. HepG2 (ATCC HB-8065), HepG2/CD8138 (link) and Hepa1-6 cells (ATCC CRL-1830) were cultured at 37°C under 5% CO2 in DMEM supplemented with 10% fetal calf serum and antibiotics (Life Technologies), as described.7 (link) HepG2 and HepG2/CD81 were cultured in culture dishes coated with rat tail collagen I (Becton-Dickinson).
Anopheles stephensi mosquitoes were fed on P. berghei or P. yoelii-infected mice using standard methods,72 (link) and kept at 21°C and 24°C, respectively. P. berghei and P. yoelii sporozoites were collected from the salivary glands of infected mosquitoes 21–28 or 14–18 days post-feeding, respectively. P. berghei and P. yoelii sporozoite infections were performed in female C57BL/6 or BALB/c mice, respectively (6 weeks old, from Janvier Labs), by intravenous injection in a tail vein. HepG2 (ATCC HB-8065), HepG2/CD8138 (link) and Hepa1-6 cells (ATCC CRL-1830) were cultured at 37°C under 5% CO2 in DMEM supplemented with 10% fetal calf serum and antibiotics (Life Technologies), as described.7 (link) HepG2 and HepG2/CD81 were cultured in culture dishes coated with rat tail collagen I (Becton-Dickinson).
5
Fibronectin, Collagen, and Integrin Protein Purification
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Fibronectin was purified from bovine plasma as described previously [25 (link)], rat tail collagen I was purchased from Becton Dickinson (Pont-de-Claix, France), vitronectin from Life Science Invitrogen (Saint-Aubin, France) and fibrinogen from EMD Millipore (Molsheim, France). Poly-lysine was obtained from Sigma-Aldrich (L'isle-d'Abeau, France). Monoclonal antibodies raised against Kindlin-2 (clone 3A3) and talin (clone TA205) were purchased from EMD-Millipore. β3 integrin mAb was obtained from Emfret (Eibelstadt, Germany). Various Alexa-488 conjugated antibodies were obtained from Invitrogen and HRP-coupled antibodies from Biorad (Marnes-la-Coquette, France). Blebbistatin was purchased from EMD Millipore, TRITC-phalloidin and Thrombin from Sigma-Aldrich and collagen for platelet aggregation assays from Helena Biosciences.
6
Quantifying Cellular Invasion Dynamics
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To assess cellular invasion in response to experimental manipulations, naïve A549 cells (15,000 cells/insert) were seeded in serum-free medium on Transwell inserts (6.5 mm, 8 μm pores, Costar) coated with 50 μg/ml rat tail collagen I (Becton Dickinson). CM from A549 cells first transfected with control or LOX siRNA and then treated with 0 or 5 Gy IR was used to attract migrating cells. For CM preparation, after irradiation, the same number of cells from each condition was seeded in serum-containing medium, and CM was collected after 24 hours. Cells seeded in inserts were allowed to migrate for 24 hours. For quantification, cells from the upper side of the insert were scraped away with cotton swabs, and then inserts were fixed in 75% methanol/25% acetic acid (v/v) and stained with DAPI. Invaded cells were counted manually under a fluorescent microscope.
7
Rodent Malaria Parasite Culture
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We used GFP-expressing P. berghei (PbGFP, ANKA strain) and P. yoelii (PyGFP, 17XNL strain) parasite lines, obtained after integration of a GFP expression cassette at the dispensable p230p locus [19 ]. PbGFP and PyGFP blood stage parasites were propagated in female Swiss mice (6–8 weeks old, from Janvier Labs). Anopheles stephensi mosquitoes were fed on PyGFP or PbGFP-infected mice using standard methods [20 ], and kept at 24°C and 21°C, respectively. PyGFP and PbGFP sporozoites were collected from the salivary glands of infected mosquitoes 14–18 or 21–28 days post-feeding, respectively. HepG2 (ATCC HB-8065), HepG2/CD81 [14 (link)] and Hepa1-6 cells (ATCC CRL-1830) were cultured at 37°C under 5% CO2 in DMEM supplemented with 10% fetal calf serum, L-glutamine and antibiotics (Life Technologies), as described [16 (link)]. HepG2 and HepG2/CD81 were cultured in culture dishes coated with rat tail collagen I (Becton Dickinson, Le Pont de Claix, France).
8
Culturing HepG2 Cells in DMEM
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HepG2 cells (ATCC HB-8065) were cultured in DMEM supplemented with 10% fetal calf serum, 1% Penicillin-Streptomycin and 1% L-Glutamine as previously described [46 (link)], in culture dishes coated with rat tail collagen I (Becton-Dickinson).
9
Isolation and Culture of Murine Hepatocytes
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Hepatocytes were isolated from 8-week-old C57Bl/6 or C57Bl/6 NOX1−/− mice according to previously described methods [17 (link)]. Briefly, mice were anesthetized with isoflurane and the portal vein was exposed and cannulated with a 26G catheter. The liver was perfused and digested with collagenase type IV (Sigma). Hepatocytes were then purified via Percoll centrifugation and seeded at a density of 1.5 × 105 hepatocytes per well onto 48-well plates coated with 0.17 mg/ml rat tail Collagen-1 (BD Biosciences). Hepatocytes were cultured in media containing DMEM with high glucose (4.5 g/L), 10% (v/v) fetal bovine serum (Biowest), 0.04 µg/ml dexamethasone, 7 ng/ml glucagon, 1% ITS+culture supplement (Corning), 1.5% 1 M HEPES, and 1% penicillin-streptomycin. The next day, hepatocytes were infected. Replicate experiments were performed with both male and female mice.
10
Silanized Glass Slide Functionalization
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