Dissociated hippocampal cultures were prepared as previous described (Kavalali et al., 1999 (link)). Sex was not determined before culturing, and all cultures consisted of neurons from mixed sexes. Whole hippocampi were dissected from PN1-3 rats, trypsinized (~4 mg/mL, Sigma), treated with DNase I, mechanically dissociated, and plated on matrigel (BD Biosciences) coated plastic or glass coverslips. Neurons were plated in MEM (no phenol red) containing 27.8 mM of Glucose, 2.4 mM of NaHCO3, 1.3 μM of Transferrin (Calbiochem), 2 mM of L-Glutamine, 4.4 μM of insulin, and 10% FBS. On DIV1, FBS concentration was reduced to 5%, L-Glutamine concentration was reduced to 500 μM, and 1x B-27 supplement (GIBCO) and 4μM of cytosine arabinoside (ARAC; Sigma) were added. On DIV4 the concentration of ARAC was reduced to 2μM. For imaging experiments, cells were also infected on DIV4 with 100 μL of lentivirus carrying either a GCaMP6s or GCaMP6s-PSD95 expression plasmid. Cells were maintained at 37°C in a 5% CO2 atmosphere without disruption following DIV4 until experiments were performed (DIV14-21). Sample size was not predetermined using statistical methods prior to experimentation. Sample sizes were based on previous studies in the field of molecular & cellular neuroscience.
Trypsinized
Manufactured by Merck Group
Sourced in United States
Trypsinized is a lab equipment product used for the enzymatic dissociation of adherent cells. It contains the protease trypsin, which breaks down the proteins that hold cells together, allowing them to detach from surfaces. This process is commonly used in cell culture applications to passage or harvest cells.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Matrigel, by BD (2 mentions)
Cytosine arabinoside arac, by Merck Group (2 mentions)
Dnase 1, by BD (2 mentions)
Aria fusion, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Live dead blue stain, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Laminin, by Merck Group (1 mentions)
Penicillin streptomycin neomycin, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Transferrin, by AppliChem (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Cmfda, by Thermo Fisher Scientific (1 mentions)
G418 selection, by InvivoGen (1 mentions)
Sep pak vac c18 cartridge, by Waters Corporation (1 mentions)
Protein assay reagent kit, by Bio-Rad (1 mentions)
Itraq labeling, by Thermo Fisher Scientific (1 mentions)
16 protocols using trypsinized
1
Dissociated Hippocampal Neuron Culture Protocol
2
Apoptosis Assay for NPC01 Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell lines were treated with different concentrations of NPC01 (0, 50, 100, and 200 mg/kg). After 24 h, the cells were trypsinized (Sigma) and centrifuged at 1000g. The pellets were washed twice using phosphate-buffered saline (PBS). Subsequently, the cells were resuspended and labeled using an Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) cell apoptosis detection kit according to the manufacturer's protocol (BD Biosciences, NJ, USA).
3
Preparation of Platelet-Rich Plasma with Bone Marrow Stromal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PRP was prepared, according to a method described by Landesberg et al (19 (link)) with modifications. Briefly, under general anesthesia with pentobarbitone sodium (30 mg/kg; Sigma-Aldrich), 6 ml fresh blood was obtained from the central artery of the rabbits ear using a syringe containing 0.6 ml acid citrate dextrose-A solution (Kermel Chemical Reagent Co., Ltd., Tianjin, China) as a anticoagulant. Subsequently, 0.2 ml whole blood was obtained to perform a platelet count. The obtained whole blood was primarily centrifuged by a centrifuge (SC-04; Zhongke Meiling Cryogenics Co., Ltd., Hefei, China) at 200 g for 10 min. Subsequently, the plasma fraction was collected and further centrifuged at 1,000 g for another 10 min. The upper three-quarters of supernatant plasma was carefully removed, and the remaining plasma and platelets were gently agitated and designated as PRP (~0.6 ml). Subsequently, 0.2 ml of the prepared PRP was drawn for platelet counting, and the remaining PRP (~0.4 ml) was retained for further use. To prepare the PRP-BMSC mixture, the BMSCs were trypsinized (Sigma-Aldrich) and diluted in PBS to 1×106 cells/ml, and 0.2 ml volumes of cell suspension were carefully mixed with the previously prepared PRP (~0.4 ml).
4
SARS-CoV-2 Nucleocapsid Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H1299-E3 cells were plated at 60,000 cells per well in 6-well plates (Corning) 1 day pre-infection. The next day the cells were infected at 1000 focus-forming units in 1 ml growth media per well. Cell–virus mixtures were incubated for 1 h at 37°C, 5 per cent CO2 then an additional 1 ml of growth media was added. Twenty-four hours post-infection, cells were trypsinized (Sigma-Aldrich), collected, and stained with Blue Live/Dead stain as per manufacturer instructions (L34961, ThermosScientific). The samples were then washed in 1 ml PBS−/− and resuspended in Cytofix/Cytoperm (BD Biosciences) for 20 min at 4˚C in the dark. The samples were then stained with 0.5 μg/ml anti-SARS-CoV-2 nucleocapsid-PE (ab283244, Abcam) for 1 h at 4˚C in the dark. Cells were analysed on an Aria Fusion (BD). Data were analyzed using FlowJo and Graphpad Prism 9.4.1 software.
5
Oxymatrine-Induced Apoptosis in Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human prostate cancer cell lines were treated with different concentrations of oxymatrine (0, 4 and 8 mg/ml). Following treatment with oxymatrine for 48 h, cells were trypsinized (Sigma-Aldrich) and centrifuged at 1,000 x g and the pellet was washed twice using PBS. Cells were resuspended and washed with PBS three times. Apoptotic cells were detected using an annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/IP) cell apoptosis detection kit, according to the manufacturer’s instructions (BD Biosciences, Franklin Lakes, NJ, USA).
6
SARS-CoV-2 Propagation and Passage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All work with live virus was performed in Biosafety Level 3 containment using protocols for SARS-CoV-2 approved by the Africa Health Research Institute Biosafety Committee. ACE2-expressing H1299-E3 cells were seeded at 4.5 × 105 cells in a 6 well plate well and incubated for 18–20 h. After one Dulbecco’s phosphate-buffered saline (DPBS) wash, the sub-confluent cell monolayer was inoculated with 500 μL universal transport medium from swabs diluted 1:1 with growth medium filtered through a 0.45-μm filter. Cells were incubated for 1 h. Wells were then filled with 3 mL complete growth medium. After 4 days of infection (completion of passage 1 (P1)), cells were trypsinized (Sigma-Aldrich), centrifuged at 300 rcf for 3 min and resuspended in 4 mL growth medium. Then all infected cells were added to Vero E6 cells that had been seeded at 1.5 × 105 cells per mL, 20 mL total, 18–20 h earlier in a T75 flask for cellto-cell infection. The coculture of ACE2-expressing H1299-E3 and Vero E6 cells was incubated for 1 h and the flask was filled with 20 mL of complete growth medium and incubated for 4 days. The viral supernatant from this culture (passage 2 (P2) stock) was used for experiments.
7
Isolation and Culture of Primary Cortical Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary cortical neurons were prepared from embryonic day 18 (E18) rats as described previously14 (link),47 (link). In brief, dissected embryonic cortices were trypsinized (Sigma-Aldrich) at 37 °C for 12 min, triturated, and seeded in 24-well plates pre-coated with poly-l -ornithine and laminin (both Sigma-Aldrich). In all, 4 × 105 cortical neurons per well were cultured in cortex medium composed of serum-free neurobasal medium supplemented with B-27, l -glutamine, penicillin/streptomycin/neomycin (all ThermoFisher Scientific), and transferrin (AppliChem) at 37 °C and 5% CO2. On day in vitro (DIV) 1, the cells were transduced with AAV.CTRL (5 × 106 transducing units (TU)) or AAV.ULK1.DN (9 × 106 TU), resulting in only minor toxicity and equal transduction rates (70–80%). Medium changes were then performed every other day. In order to induce autophagy in selected conditions, rapamycin (750 nM, Sigma-Aldrich) was added to the medium 24 h before lysis.
8
Adipocyte-Macrophage Phagocytosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary adipocytes (PA) differentiated for ten days and SGBS adipocytes (SA) were stained with Hoechst 33342 (Sigma, 50 μg/ml) and 1 μg/ml Nile red for 30 min. To decrease nonspecific accumulation of Hoechst and Nile red by macrophages during the phagocytosis process, cells were carefully washed two times in PBS. Macrophages were stained with fluorescent cell tracer green CMFDA (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Macrophages were layered on the top of adipocytes in a ratio of 5 : 1 and were cocultured for 24 h at 37 °C in a 5% CO2 atmosphere. Phagocytic ratio was determined counting the macrophages containing lipid droplets applying laser-scanning cytometry (LSC). For flow cytometric measurements, cells were trypsinized (Sigma) and centrifuged at 1800 rpm for 10 min; when macrophages sedimented to a pellet, whereas adipocytes, owing to their lipid content, remained in the supernatant. Cells in the pellet were examined by a FACSCalibur BD flow cytometer and list mode data were analyzed by WinMDI2.8 software (Freeware, written by Josef Trotter, downloaded from http://facs.scripps.edu ).
9
HEK-293 Cells Stable Transfection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293 cells were transfected with the plasmid pEntry-hscFSH, previously digested with AgeI (New England Biolabs, USA). Transfection was performed in 100 mm plates using the polyethylenimine-based method mentioned above. Two days after transfecting with 20 µg of DNA per plate, the cells were trypsinized (Sigma, USA) and plated for G418 selection (500 μg/ml) (InvivoGen, USA). Fresh G418-containing medium was added every 4 days. Colonies resistant to G418 were visible after 20 days. Fluorescence was observed in dark fields using an excitation filter of 482/18 nm and an emission filter of 532/59 nm. The size of clones was measured, and the fluorescence intensity was quantified using the Photoshop program as described below.
10
Proteomic Analysis of ALS Cerebrospinal Fluid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the removal of 22 high-abundance proteins, including albumin and IgG, using ProteoMiner low abundance protein enrichment kits (Bio-Rad Laboratories, Inc., Hercules, CA, USA), protein quantification was conducted using a Protein Assay reagent kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) based on Bradford methods, according to manufacturer's protocol. iTRAQ labeling was performed according to the manufacturer's protocol (Applied Biosystems Life Technologies, Foster City, CA, USA). Briefly, 100 µg CSF proteins from the ALS and NC groups were precipitated with cold acetone (ratio of acetone:sample, 5:1) for 1 h at −20°C and resuspended in 20 µl dissolution buffer, respectively. Following centrifugation at 2,000 × g for 15 min and disposal of the supernatant, the precipitant was dissolved into 20 ul iTRAQ solution and 1 ul 1% sodium dodecyl sulfate (SDS). Subsequently, 1 ul cysteine sealing reagent was added for 10 min at room temperature. Proteins were trypsinized (Sigma-Aldrich, St. Louis, MO, USA) at 37°C overnight (ratio of enzyme:protein, 1:20). Peptides were labeled with iTRAQ regents for 1 h at room temperature. iTRAQ regents 113 and 118 were used to label the peptides from the NC and ALS groups, respectively. Following this, samples were mixed, desalted with Sep-Pak Vac C18 cartridges (Waters Corporation, Milford, MA, USA) and dried in a vacuum concentrator.
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!