THP-1 macrophages for RNA sequencing analysis were treated for 15 h with PyV-derived VLPs (20 µg/ml). After the treatment, cells were washed with PBS and detached with TrypLE reagent after 15 min of incubation at a cell culture incubator (37°C, 5% CO2). After centrifugation, cells were washed twice with 1× RNase-free PBS (cat#AM9624, Invitrogen). After final centrifugation, cells were resuspended in RNase-free PBS and kept on ice till cell preparation for RNA sequencing analysis
Am9624
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AM9624 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. The core function of the AM9624 is to provide a controlled environment for various laboratory applications. Detailed specifications and intended use are not available in this unbiased, factual description.
Automatically generated - may contain errors
Lab products found in correlation
S7903, by Merck Group (1 mentions)
M2 media, by Merck Group (1 mentions)
Ultrapure distilled water, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting media, by Vector Laboratories (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
T2600000, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Yeasen (1 mentions)
9 protocols using am9624
1
THP-1 Macrophage RNA-Seq Analysis
2
Embryo Fixation and Cryosectioning
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Embryos were dissected from the uteri, washed in M2 medium (Sigma Aldrich, 7167) and immediately placed in 4% paraformaldehyde (PFA) (Thermo Scientific, 28908) in 1× PBS (Invitrogen, AM9624) for 30 min at room temperature. The embryos were then washed and immersed in 30% RNase-free sucrose (Sigma Aldrich, 84097) in 1× PBS at 4 °C until the embryo sank to the bottom of the tube. Afterwards, each embryo was positioned in a sagittal orientation in a tissue base mold (Sakura, 4162) in optimal cutting temperature (OCT) compound solution (Sakura, 4583), frozen in dry ice isopropanol (VWR, 20842) and stored at −80 °C. Tissue sections (20 µm) were cut using a cryotome, collected on the functionalized coverslips and stored at −80 °C.
3
Lung Tissue Preparation for Downstream Analysis
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Mice were injected with a mix of 100 mg/kg ketamine and 10 mg/kg xylazine and we waited for the mice to be fully asleep. Then, the ribcage was opened, the heart was perfused with 10 ml of cold 1× PBS pH 7.4 (Invitrogen, AM9624) and it was resected just after the perfusion was finished. Then, the mouse trachea was perfused with cold 4% paraformaldehyde (PFA) (Euromedex, 15714-S) until the lungs were fully expanded with no air left inside. The trachea was closed with a thread to avoid PFA leakage. The lungs were resected out of the ribcage and kept in a falcon with cold 4% PFA overnight (o/n) under rotation at 4 °C. After fixation, the 5 lobes were separated and kept individually in cold 1× PBS containing 30% sucrose (Sigma, S7903) during 6 h under rotation at 4 °C. Lobes were rinsed in cold 1× PBS and pre-embedded in cold 50% optimal cutting temperature (OCT) compound (VWR, 411243) diluted in 1× PBS during 30 min under rotation at 4 °C. Finally, each lobe was embedded in square embedding molds (VWR, POLS18646ACODE45) containing OCT, frozen in dry ice during 20 min and stored at −80 °C.
4
PEG Hydrogel Formation for LiverChip
5
Embryo Fixation and Sectioning
6
Perfusion and Tissue Processing for Mouse Brain Analysis
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Eighteen days after LPC injection, mice were anaesthetized with an i.p. injection of MMF (fentanyl (0.05 mg/kg)–midazolam (5 mg/kg)–medetomidine (1 mg/kg)) and transcardially perfused with 2 UI/mL Heparin (Heparin-Natrium-25000-ratiopharm®, PZN: 03029843) in HBSS (no calcium, no magnesium, Gibco™, 14175129) for 3 min and 4% paraformaldehyde (PFA, EM Grade, Electron Microscopy Sciences, Cat. No. 15710, diluted in 10X PBS, Invitrogen™ (to a final concentration 1x), AM9624 and UltraPure™ Distilled Water, Invitrogen™, 10977-035) for 5 min, before carefully removing the brain from the skull. Afterwards, the brains were fixed by submerging in 4% PFA for 6 h, followed by 14 h in 15% sucrose (Sigma-Aldrich, S0389 in UltraPure™ Distilled Water, Invitrogen™, 10977-035) and 5 h in 30% sucrose. Next, the PFA-fixed brains were simultaneously embedded in Tissue-Tek® O.C.T.™ Compound (Sakura, 4583) and frozen in a plastic mold on dry ice. For the “standard protocol”, fresh frozen brain samples were used: the mouse was only perfused with Heparin in HBSS and directly embedded and frozen in Tissue-Tek® O.C.T.™ Compound on dry ice. Brains were stored at −80 °C until further processing.
7
Fixation and Staining of C. elegans
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Stage-synchronized worms were harvested with M9 buffer with polyethylene glycol 4000 (PEG 4000) (0.01%; Tokyo Chemical Industry, Japan) and washed three times with M9 buffer. Worms were fixed with 4% paraformaldehyde (158127, Sigma-Aldrich, MO, USA) solution in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4; AM9624, Thermo Fisher Scientific, MA, USA) for 45 min with gentle agitation. After fixation, worms were washed with PBS containing 0.01% PEG 4000 twice with gentle agitation for 5 min for each washing step. Fixed worms were stored in 70% ethanol at 4°C overnight. Worms were placed on a 2% agarose pad on a slide glass, and the worms were soaked in a drop of 4′,6-diamidino-2-phenylindole (DAPI) solution (2 ng/μl) (62248, Thermo Fisher Scientific, MA, USA) in VECTASHIELD antifade mounting media (Vector Laboratories, CA, USA). After covering the agar pad with a coverslip, worms were incubated for at least 30 min in the dark before imaging.
8
Single-molecule Fluorescence Imaging Setup
9
Quantifying Apoptosis in PC-12 Cells
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The apoptosis of PC-12 cells was measured by Annexin V-FITC/PI apoptosis detection kit (40302ES20, Yeasen, Shanghai, China). After transfection and LPS treatment, PC-12 cells were digested in EDTA-free trypsin (T2600000, Sigma-Aldrich, USA), and washed with phosphate buffered saline (PBS; AM9624, Thermo Fisher, USA) thrice. The cells were then resuspended by 1× Binding Buffer to be 1 × 106 cells/mL and added with Annexin V-FITC solution (5 μL) and propidium iodide (PI) solution (10 μL). After 10 min incubation in the dark, apoptotic cells were examined by a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA).
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