α-, β- and γ-CD, pepsin-HCl, pancreatine and bilis were purchased from Sigma-Aldrich (Madrid, Spain). Hydroxypropyl-beta- and methyl-beta-cyclodextrin (HPβ-CD DS = 5 and Mβ-CD DS = 5.4) were purchased from Carbosynth (Berkshire, UK). Roflumilast (CID 5281717) was purchased from Xi An Kerui Biochemical CO (Xi’an, China) and used as received. The samples were stored in darkness. Ethanol (absolute, analysis grade) was purchased from Panreac (Madrid, Spain).
Pancreatine
Manufactured by Merck Group
Sourced in Spain
Pancreatine is a laboratory enzyme product produced by Merck Group. It is a mixture of digestive enzymes derived from the pancreas of animals. The core function of Pancreatine is to facilitate the breakdown and digestion of macromolecules such as proteins, fats, and carbohydrates in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Pepsin hcl, by Merck Group (1 mentions)
Cobalt chloride, by Merck Group (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Phosphate buffer solution ph 8, by Merck Group (1 mentions)
Pepsine, by Merck Group (1 mentions)
Oxgall bile, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
Eugenol, by Merck Group (1 mentions)
Hydrochloridric acid, by Merck Group (1 mentions)
Bile extract, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Carvacrol, by Merck Group (1 mentions)
5 protocols using pancreatine
1
Cyclodextrin-Assisted Formulation Development
2
Isolation of neonatal mouse cardiomyocytes and SARS-CoV-2 infection assay
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Neonatal mouse ventricular cardiomyocytes were isolated on the day of birth (P0). Hearts were isolated and atria removed. Next, ventricles were mechanically minced before being put on multiple enzymatic digestion steps in digestion buffer [Pancreatine (Sigma), Collagenase type 2 (Gibco) and Dnase 1 (Invitrogen)]. After the final digestion step, cells were filtered through a 100 μM cell strainer and pre-plated for 1 h at 37°C, allowing non-cardiomyocytes to adhere. Finally, the medium containing the cardiomyocytes was collected and after centrifugation, cardiomyocytes were plated in fresh medium on gelatin-coated 24-well plates. Vero E6 cells (African green monkey kidney, ATCC CRL-1586) were cultured in minimal essential medium (15188319, Gibco) supplemented with 10% fetal bovine serum, 1% L-glutamine (25030149, Gibco), and 1% bicarbonate (25080094, Gibco). Cells were infected with SARS-CoV-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 407976|2020-02-03)] at a multiplicity of 0.2 TCID50 per cell for 20 h and subsequently treated with 800μM Cobalt Chloride (CoCl2, C-8661, Sigma) for 6 h. Cells were fixed in 1% PFA overnight and stained with HIF1α (1/50) and Topro (1/1,000).
3
Simulating Gastrointestinal Digestion of Probiotics
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In order to simulate the effect of the gastrointestinal digestion on the probiotic microorganisms, the methodology described in [17 ] was followed. Ti referred to a L. salivarius spp. salivarius content (CFU/g), meanwhile ti referred to a moment in the gastrointestinal digestion. Ten grams of the sample were mixed with 10 mL of pepsine 0.6% (w/v) (Sigma-Aldrich, Steinheim, Germany) adjusted to pH 3 with HCl 4M (t1—T1) and stirred at 37 °C for 90 min (t2—T2). The addition on a phosphate buffer solution (pH 8) with 10% of bile (Sigma-Aldrich, Steinheim, Germany) was performed in the third step (t3—T3). The phosphate buffer solution with 0.3% of bile and 0.1% pancreatine (Sigma-Aldrich, Steinheim, Germany) was added on following an incubation at 37 °C for 90 min (t4—T4). The results provided are the average of four replicates.
4
Evaluating L. crispatus Survival in GI Tract
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Shake flask experiments were performed to evaluate the capability of L. crispatus L1 to survive the gastrointestinal tract. Simulated gastric and pancreatic juices were prepared by slightly modifying the protocols reported by Kos and colleagues [42 ]. Briefly, gastric juices were simulated with a solution of NaCl, 125 mM, KCl 7 mM, NaHCO3, 45 mM and pepsine (Sigma Aldrich) 0.3% (w/v), with a final pH equal to 2 obtained by HCl addition. Either 6.0∙108 cells · ml−1 (low dose, minimal starting density for shake flasks experiments necessary to avoid the lag phase) or 1.8·109 cells · ml−1 (high dose, typical amount delivered in probiotic commercial products) were inoculated into the medium and incubated 2–3 h in shaker at 37°C and 110 rpm to simulate physiological conditions. This step was followed by centrifugation (15 min at 1200 × g) and re-suspension of the cells in a solution containing pancreatine (Sigma Aldrich) 0.1% (w/v), Oxgall bile (Sigma Aldrich) 0.15% (w/v) with a final pH equal to 4, to simulate pancreatic juices. The suspension was incubated for 3 h, after which cells were centrifuged and re-suspended in fresh MRS medium to evaluate bacterial growth. At the end of each step cell viability was measured by plating aliquotes and counting colony forming units (cfu).
5
Synthesis of Antimicrobial Silica Microparticles
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Triethanolamine (TEAH3), sodium hydroxide (NaOH), tetraethylorthosilicate (TEOS), Ncetyltrimethylammonium bromide (CTAB), paraformaldehyde, (3-aminopropyl) triethoxysilane (APTES), carvacrol (98% w/w), eugenol (99% w/w), hydrochloridric acid (HCl), pepsin, bile extract and pancreatine (all three of porcine origin), and all the other reagents used to prepare SID solutions and ALF, were supplied by Sigma-Aldrich (Madrid, Spain). Vanillin was acquired from Ventós (Barcelona, Spain). Amorphous silica microparticles (SYLYSIA® SY350/FCP; 4 (0.1) μm) were purchased from Silysiamont (Milano, Italy). All the employed materials were of standard analytical grade.
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