Proliferation was assessed by DNA quantification. At each time interval, the constructs were washed in PBS 1x (Gibco™, Waltham, MA, USA) and stored frozen at −80 °C until analysis. For DNA quantification, the constructs were digested in 20 UI/mL Papain solution (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 65 °C for 1 h and total DNA (ng DNA/mg construct) was determined using a Quant-iT Pico Green® dsDNA kit (P7581; InvitrogenTM; Waltham, MA, USA), following the manufacturer’s instructions.
Papain solution
Manufactured by Worthington
Sourced in United States
Papain solution is a liquid product containing the enzyme papain, derived from the fruit of the papaya plant. Papain is a proteolytic enzyme that can break down proteins. The solution is intended for laboratory and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Poly d lysine, by Merck Group (12 mentions)
Glutamax, by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
L glutamine, by Thermo Fisher Scientific (8 mentions)
Neurobasal medium, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Fura 2 am, by Thermo Fisher Scientific (4 mentions)
Annexin 5, by BD (4 mentions)
Poly d lysine, by BD (4 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Neurobasal a, by Thermo Fisher Scientific (3 mentions)
Mouse anti thy1.1 antibody, by Abcam (3 mentions)
Fetal bovine serum (fbs), by Lonza (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Countess 2, by Thermo Fisher Scientific (3 mentions)
Cmf hbss, by Thermo Fisher Scientific (2 mentions)
Dnase 1 solution, by Merck Group (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
59 protocols using papain solution
1
DNA Quantification for Cell Proliferation
2
Bee Brain Dissection and Dissociation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before dissection, all bees were anesthetized by carbon dioxide. The whole brains of alive bees were dissected in ice-cold SF9-III medium for each biological replicate and immediately transferred to a pre-cooling tube containing SF9-III medium. Four to five brains were pooled. The dissociated brains were incubated with papain solution (Worthington, 1 μg/μL) at 35°C for 30 min, and briefly centrifuged (50 g, 10 s) to collect cell suspension and remove undigested tissue fragments. The cell suspension was centrifuged again (300 g, 5 min), and the pellet was washed twice with DPBS. Subsequently, the cell pellet was resuspended into 3 mL PBSB (DPBS+0.04% BSA) and filtered through a 20 μm pluriStrainer. An AO/PI Dual-fluoresces counting assay performed on the Countstar FL system detected cell counting and viability. The cell suspension (cell viability >95%) was adjusted to a final concentration of 700∼800 cells/μL.
3
Isolation and Culture of Murine Motor Cortical Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cerebral motor cortices of mice were isolated, and cells were incubated as described previously [18 (link)]. Six-well cell culture plates (SPL) pre-coated with 0.1 mg/ml of PDL (Sigma) were incubated overnight at 37 °C, followed by two washings with distilled water (Sigma). Motor cortices of non-TG or TG mice at P3 were collected using fine forceps (FST) into dissociation solution (DS) containing magnesium chloride (Sigma), Hepes (Gibco), sodium sulfate (Sigma), potassium sulfate (Sigma), kyneuric acid (Sigma), glucose (Sigma), APV (Sigma), penicillin/streptomycin (P/S) (Gibco), and B27 (Gibco). Cells were then dissociated in a papain solution (Worthington Biochem) for 15 min, and incubated in inhibitor solution containing ovomucoid (Sigma) for 1 min. After being washed with Opti-MEM (Gibco) solution containing APV (Sigma) and B27, 3.2 × 105 cells were seeded with serum-free media (SFM) containing BSA, L-glutamine (Sigma), P/S, glucose, and B27 in neurobasal media (Gibco). Cells were then treated with inhibitors for Met (PHA665752), ERK (U0126), PI3K (LY294002), p38 (SB203580), and JNK (SP600125). Thirty minutes later, cells were cultured in the presence of 100 ng/ml of rHGF (R&D Systems). 3 days later, cells were subjected to immunocytochemistry (ICC) assay.
4
Isolation of Neuronal Mitochondria and Cytosol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, the cell suspension was prepared according to a previously described method97 (link). Briefly, brain tissues were dissociated with 300 μL papain solution (28 units/mL, Worthington) containing 1% DNase and 12 mg/mL L-cysteine in DMEM/F12. The mixture was incubated at 37 °C for 15 min with gentle agitation. Dissociated cells were then washed twice with washing buffer (100 mL: 650 μL 45% glucose, 500 μL 1 M HEPES and 5 mL FBS into 93.85 mL 1×DPBS). Subsequently, a mitochondria isolation kit for tissue (Abcam, #ab110168) was used to obtain the cytoplasmic and mitochondrial fractions according to the manufacturer’s instructions with minor changes. Neuronal cells were homogenized in ice-cold mitochondria isolation buffer containing protease inhibitor cocktail after resuspension and then centrifuged at 1000 × g for 10 min twice at 4 °C to remove debris. The supernatant was centrifuged at 12,000 × g for 15 min twice to isolate the mitochondrial fraction. Finally, the resulting pellet containing mitochondria was washed twice with isolation buffer, and the remaining supernatant served as the cytosolic fraction.
5
Culturing Hippocampal Neurons from FVB/NJ Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hippocampal cultures from FVB/NJ mice were established from postnatal day 0–2 pups and maintained in culture as described previously (Chernyuk et al., 2019 (link), Popugaeva et al., 2015 (link), Sun et al., 2014 (link)).
Briefly, hippocampus was dissected in sterile ice-cold dissection buffer (1% 10x CMF-HBSS (Gibco, #14185), 1% Pen Strep (Gibco, #15140), 1.6 мМ HEPES (Sigma, #H3375), 1 мМ NaHCO3 (Sigma, #S5761); pH = 7.6). After that it was digested with papain solution (Worthington, #LK003176) 30 min at 37 °C, then twice triturated with 5 mg/ml DNase I solution (Sigma, #DN-25). Neurons were plated in 24-well culture plate on 12 mm glass coverslips (Thermo Scientific, #CB00120RA1) precoated with 0.1 mg/ml poly-D -lysine (Sigma, #P0899) in Neurobasal (Gibco, #10888) medium supplemented with 1% FBS (Gibco, #10500), 2% 50xB27 (Gibco, #17504), 0.05 мМ L- Glutamine (Gibco, #250030) and maintained at 37 °C in a 5% CO2 incubator. A half of the medium was replaced with a new culture medium at DIV 7 and DIV 14.
Briefly, hippocampus was dissected in sterile ice-cold dissection buffer (1% 10x CMF-HBSS (Gibco, #14185), 1% Pen Strep (Gibco, #15140), 1.6 мМ HEPES (Sigma, #H3375), 1 мМ NaHCO3 (Sigma, #S5761); pH = 7.6). After that it was digested with papain solution (Worthington, #LK003176) 30 min at 37 °C, then twice triturated with 5 mg/ml DNase I solution (Sigma, #DN-25). Neurons were plated in 24-well culture plate on 12 mm glass coverslips (Thermo Scientific, #CB00120RA1) precoated with 0.1 mg/ml poly-
6
Newborn Rat Pup Neuronal Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wistar rat pups (newborn P0–1) were obtained from the Valladolid University animal facility. All animals were handled according to the ethical standard of Valladolid University under protocols approved by the animal housing facility and in accordance with the European Convention 123/Council of Europe and Directive 86/609/EEC. Fura-2/AM and Rhod-5N are from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) is from Lonza (Barcelona, Spain). Horse serum, neurobasal medium, HBSS medium, B27, L-glutamine and gentamycin are from Gibco (Barcelona, Spain). Papain solution is from Worthington (Lakewood, NJ, USA). The poly-D-lysine and annexin V are from BD (Madrid, Spain). DNase I and antibody against the MCU are from Sigma (Madrid, Spain). IP3R1 and IP3R2 primary antibodies are from Santa Cruz Biotechnology (Dallas, TX, USA). IP3R3 primary antibody is from BD Transduction Laboratories (Madrid, Spain). ER tracker, mitotracker, tetramethylrhodamine, methyl ester (TMRM) and CM-H2DCFDA are from ThermoFisher Scientific. Aβ1–42 peptide is from Bachem AG (Bubenforf, Switzerland). Other reagents and chemicals were obtained either from Sigma or Merck.
7
Primary Hippocampal Culture Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hippocampal cultures were prepared by dissecting both hippocampi of P0-P1 C57BL/6JOlaHsd (α-Syn−/−) mouse brains, as described previously (80 (link), 81 (link)). After trituration in 20 units/ml of papain solution (Worthington Lakewood, NJ, USA), 1 × 105 cells were plated on coverslips that were pre-coated with 5 μg/ml of poly-D-lysine (Sigma-Aldrich, Rehovot, Israel) in a 24-well–plate containing 1.5 ml of Neurobasal-A medium (Gibco, ThermoFisher Scientific, Petah Tikva, Israel) supplemented with 5% fetal bovine serum, 2% B-27 (Gibco, ThermoFisher Scientific), 1% Glutamax I, and 1 μg/ml of gentamicin. After 1 day, the solution was replaced to 1 ml of Neurobasal-A supplemented with 2% B-27 and 1% Glutamax I. To slow the proliferation of glial cells, 1 μm cytosine β-d -arabinofuranoside (Ara-C; Sigma-Aldrich) was added to the culture at 2 DIV. Cultures were maintained at 37 °C in a 5% CO2 humidified incubator until used at 8-13 DIV as indicated. For measurements of Tf-568 endocytosis, neurons were prepared and grown as previously describe (5 (link)).
8
Embryonic Rat Cortical Neuron Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CTX were prepared from embryonic day 17‐18 (E17‐E18) Sprague Dawley rat embryos, as described previously.21 Briefly, dissected cerebral cortical tissue was collected into ice‐cold Hank's Balanced Salt Solution without Ca2+ or Mg2+, and then dissociated in papain solution for 40 minutes at 37°C following the manufacturer's instructions (Worthington Biochemical, Lakewood, US). Cortical cells were plated on poly‐L‐ornithine coated 96‐well plates at a density of 20 000 cells/well in 125 μL Neurobasal medium containing 2% B27 supplement, 0.5 mmol/L glutamax, 1% FBS and 100 U/mL penicillin/streptomycin. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. A further 125 μL Neurobasal medium containing 2% B27, 0.5 mmol/L glutamax was added on DIV 4. Cells were maintained by replacement of half the medium twice per week. On DIV 11, 1.5 μmol/L cytosine β‐D‐arabinofuranoside was added to the medium to prevent proliferation of non‐neuronal cells.
9
Isolation of Retinal Ganglion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary RGCs were isolated according to the two-step immunopanning protocol as described previously [19 (link)]. Briefly, the retinas were incubated in papain solution (16.5 U/mL; Worthington Biochemical Corp., Lakewood, NJ, USA) for 30 min to get the single cell suspension. Macrophages and endothelial cells were then removed from this suspension by panning with the anti-macrophage antibody (AIA31240, Accurate Chemical, Carle Place, NY, USA). Next, RGCs were bound to the panning Petri dish containing the antibody against CD90.2/Thy1.2 and were then released by trypsin incubation. The two-step immunopanning protocol takes a few hours to isolate the RGCs, which may affect RNA-seq data. However, previous studies have shown that observed changes are minor, affecting mostly the activity of immediate early genes [59 (link),60 (link),61 (link)].
10
Isolation and Purification of Retinal EGFP Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!