THP-1 cells were seeded on glass coverslips and differentiated into macrophages overnight, before fixation with 4% formaldehyde (FA; Sigma-Aldrich, F1635) containing 0.2% Triton X-100 (Sigma-Aldrich, T9284) for 10 minutes and permeabilized with 1% Triton X-100 for 10 minutes. Fixed cells were washed with PBS and non-specific binding was blocked with 0.5% milk in PBS before incubation with primary antibodies in 0.25% milk in PBS for 1hr. Unbound antibody was removed by washing with PBS and secondary antibody in 0.25% milk in PBS was added and incubated for 30 minutes. Unbound secondary antibody was removed and cells were washed with PBS and stained with DAPI (Sigma-Aldrich, D9542) for 1 minute. Coverslips were washed and mounted with Mowiol mountant (Sigma-Aldrich, 324590). Fluorescence was visualized with a DeltaVision Spectris Deconvolution Microscope (GE Healthcare Life Sciences) with a 100x PlanApo 1.35 objective (Olympus). Images were deconvolved with the SoftWoRx software (GE Healthcare Life Sciences). For the experiments studying the effect of PPAR ligands on FAMIN expression, the number of peroxisomes was calculated using a custom ImageJ macro on 10 images per condition and triplicate experiments. Values were normalized according to the controls (untreated cells).
F1635
Manufactured by Merck Group
Sourced in United States, Germany
The F1635 is a laboratory equipment product manufactured by Merck Group. It is a device designed for use in scientific and research settings. The core function of the F1635 is to perform specific laboratory tasks, however, a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
T9284, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Ab128245, by Abcam (2 mentions)
Plan fluor 20x objective, by Nikon (2 mentions)
G5516, by Merck Group (2 mentions)
Plan fluor 40x air objective, by Nikon (2 mentions)
Softworx software, by GE Healthcare (1 mentions)
Facscanto, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Primovert microscope, by Zeiss (1 mentions)
Axiocam erc5s camera, by Zeiss (1 mentions)
Senescence cells histochemical kit, by Merck Group (1 mentions)
H1009 100ml, by Merck Group (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein a, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Anti h3k4me3, by Merck Group (1 mentions)
Anti mre11, by Novus Biologicals (1 mentions)
25 protocols using f1635
1
Immunofluorescence Imaging of Macrophages
2
Flow Cytometry Surface Marker Analysis
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For analyzing surface markers with flow cytometry, cells were collected, washed, and stained with antibodies (Table 1 ) at 4 °C for 30 min in the dark. For intracellular staining of phospho-CD3ζ, T cells were fixed with a 1.5% formaldehyde solution (F1635; Sigma-Aldrich, St. Louis, MO, USA), washed, permeabilized with pre-chilled 100% methanol (Fisher Scientific, Pittsburgh, PA) on ice for 15 min, and then washed three times, followed by a CD247(pY142)-specific antibody (Table 1 ) stain for 60 min at room temperature, in the dark. All samples were acquired on either BD FACSCalibur or BD FACSCanto flow cytometers (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed with the FlowJo software (Tree Star Inc., Ashland, USA).
3
Quantifying Autophagic Structures in EGFP-LC3 Cells
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The stable EGFP-LC3 H1299 cell line was
used to detect autophagic structures by fluorescence spinning disk
microscopy. Cells were grown on glass coverslips, treated with compounds
for 48 h and fixed in 3.7% formaldehyde (Sigma, F1635) DNA was counterstained
with 0.05 μg/mL DAPI (4,6-diamidino-2-phenylindole; Sigma-Aldrich,
D9542). At least 500 cells were counted for each experimental point,
and cells showing ≥15 dots were considered positive. Dots were
counted using a pipeline created in a previous work on the CellProfiler
software.68 (link) Data are presented as median
with interquartile range. Samples were observed under a Nikon Eclipse
Ti2 microscope with a 60× (1.4 NA) objective, equipped with a
CrestOptics X-Light V3 confocal spinning disk and a back illuminated
Kinetix sCMOS camera.
used to detect autophagic structures by fluorescence spinning disk
microscopy. Cells were grown on glass coverslips, treated with compounds
for 48 h and fixed in 3.7% formaldehyde (Sigma, F1635) DNA was counterstained
with 0.05 μg/mL DAPI (4,6-diamidino-2-phenylindole; Sigma-Aldrich,
D9542). At least 500 cells were counted for each experimental point,
and cells showing ≥15 dots were considered positive. Dots were
counted using a pipeline created in a previous work on the CellProfiler
software.68 (link) Data are presented as median
with interquartile range. Samples were observed under a Nikon Eclipse
Ti2 microscope with a 60× (1.4 NA) objective, equipped with a
CrestOptics X-Light V3 confocal spinning disk and a back illuminated
Kinetix sCMOS camera.
4
Formalin-Induced Nociceptive Behavior in Mice
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For the formalin test, the mice received intraplantar injection of formalin (Sigma, F1635, 5%, vol/vol diluted in saline, 10 μl) and then replaced in transparent plastic chambers. Morphine (10 mg/kg) or saline was injected subcutaneously or intraperitoneally 30 min before formalin injection. Total duration of the animal spending in licking and flinching behaviors of the injected paw was counted manually in 5 min interval for 1 hr. The first phase of nociceptive responses was calculated during 0–10 min, and the second phase of nociceptive responses was calculated during 10–60 min.
5
Quantifying Senescence-Associated β-Galactosidase
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SAβ-gal activity was measured using a Senescence Cells Histochemical Kit (CS0030, Sigma) according to the protocol supplied by the manufacturer. Briefly, cells were incubated for 24 h with or without addition of 200, 400, and 800 μM H2O2 (H1009-100ML, Sigma) or 5 mM N-acetyl-cysteine (NAC) (A9165-25G, Sigma). After fixation with Fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 6 min, they were incubated with Staining Mixture at 37 °C overnight. Cells were examined under a ZEISS Primovert microscope (ZEISS, Germany) with ZEISS Axiocam ERc 5s camera (ZEISS, Germany). The cytosol in senescent cells was stained blue. To determine the SA-β-gal activity in uteruses, uteruses were collected from mice, fixed with 10% formaldehyde (v/v) (F1635, Sigma) for 10 min at room temperature, and incubated with Staining mixture for 24 h at 37 °C.
6
Sonication-based ChIP-seq for Testicular Cells
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Ten million bulk testicular cells were fixed in 1% formaldehyde (Sigma, F1635) at 37 °C for 10 min. Fixation was quenched with glycine (Sigma) at a final concentration of 125 mm . Cells were washed twice with cold PBS, and pellets were snap frozen on dry ice and stored at −80 °C until sonication. Sonication, immunoprecipation, and library preparation were performed as previously described in (Canela et al.16 (link)). In brief, frozen pellets were resuspended in 1 mL cold RIPA buffer (10 mm Tris-HCl pH 7.5, 1 mm ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulphate, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 Complete Mini EDTA-free proteinase inhibitor tablet (Roche)) and sonicated using a Covaris S220 at duty cycle 20%, peak incident power 175, and cycle/burst 200 for 30 min at 4 °C.
Chromatin was precleared with 40 μL prewashed Dynabeads Protein A (ThermoFisher) for 30 min at 4 °C, followed by incubation with 40 μL Dynabeads Protein A bound to either 6 μL anti-H3K4me3 (Millipore, 07–473) or 4 μL anti-MRE11 (Novus, NB100-142) overnight at 4 °C. Beads were washed and cross-linking reversed the next day as described in (Canela et al.16 (link)). Immunoprecipitated DNA was removed from beads and stored at −20 °C until library preparation, which was performed as described in (Canela et al.16 (link)).
Chromatin was precleared with 40 μL prewashed Dynabeads Protein A (ThermoFisher) for 30 min at 4 °C, followed by incubation with 40 μL Dynabeads Protein A bound to either 6 μL anti-H3K4me3 (Millipore, 07–473) or 4 μL anti-MRE11 (Novus, NB100-142) overnight at 4 °C. Beads were washed and cross-linking reversed the next day as described in (Canela et al.16 (link)). Immunoprecipitated DNA was removed from beads and stored at −20 °C until library preparation, which was performed as described in (Canela et al.16 (link)).
7
Formaldehyde Crosslinking and ChIP-seq Analysis
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Crosslinking was performed with 1% formaldehyde (Sigma, F1635) at 37°C for 10 min. A final concentration of 125 mM glycine was added for 5 minutes at RT to quench formaldehyde crosslinking. Cells were washed with ice-cold PBS, harvested on ice and then transferred to 50 mL falcon tubes. After centrifugation at 4,000 rpm for 5 min at 4°C, cell pellets were transferred to Eppendorf tubes and frozen using liquid nitrogen. The cell pellets were stored at −80°C for further analysis. Two independent clonal lines per genotype were used for ChIP-seq analysis.
8
Quantifying Nuclear β-Catenin Dynamics
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Cells were washed three times with phosphate-buffered saline (PBS), fixed with 3.7% Formalin-PBS (F1635, Sigma-Aldrich) then permeabilized with 0.2% Triton-X-100-PBS (0694, Amresco, USA) for 10 min, blocked with 3% BSA-PBS (A7906, Sigma-Aldrich) and incubated with primary antibodies for 1 h at room temperature. Cells were washed twice with PBS before secondary antibodies were added for 40 min. Cells were subsequently mounted with Vectashield (Vector Laboratories, USA) and visualized by fluorescence microscopy and imaged and scored using an Olympus FV1000 confocal laser microscope. Fluorescence recovery after photobleaching (FRAP) assay to measure nuclear import rates of β-catenin-GFP in live NIH3T3 cells was as described previously (Jamieson et al., 2011 (link)), using an Olympus FV1000 confocal laser microscope. Briefly, three pre-bleach images of the cell were acquired, then the nucleus was bleached (60–70 frames). Post-bleach imaging was in two stages: 30 frames at the fastest interval, then 30 frames at 10 s intervals. Fluorescence intensities for cytoplasm, nucleus and background were acquired using Olympus Fluoview software and exported to a Microsoft Excel file.
9
Formaldehyde Fixation for Cell Cultures
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All the cells were fixed using principally the same conventional 40 mg/ml formaldehyde in PBS solution. All the so-called flat-substrate cells were fixed using paraformaldehyde (158127, Sigma-Aldrich), the fixative was prepared as follows. 40 g of solid paraformaldehyde was dissolved in 900 ml water heated to 65 ºC and mixed for 45 min. A few drops of 1 M NaOH was added until the solution became clear. The solution was cooled and mixed with 100 ml of 10X PBS, and pH was titrated to 7.3 using 1 M NaOH or HCl. Finally, the fixative was filtered and stored in −20 ºC aliquots for later use. The cells cultured in the PLA scaffolds were fixed using more convenient 1/10 dilution of commercial 10–15% methanol-stabilized 37 wt. % formalin (F1635, Sigma-Aldrich) mixed with 1/10 10X PBS and 8/10 water. Although two variations of the principally same fixative were used, the same fixative recipe was used for all the within-experiment cells to ensure comparability.
10
Formalin-Induced Pain Modulation
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Forty microliter of formalin (2% in saline; F1635, Sigma) with PBS (“Formalin + vehicle” group), formalin with PDGFR-β-Fc (“Formalin + PDGFR-β-Fc” group), or PDGFR-β-Fc alone (in saline; “PDGFR-β-Fc alone” group) were injected into the left hind paw. In another experiment, 40 μL of formalin (2% in saline) with either saline (“Formalin alone” group) or imatinib (60 ng/g; (“Formalin + imatinib” group) were injected. For the positive control experiment, either 1.5 or 5 mg/kg of morphine were injected into the loose skin under the neck, 30 minutes before injection of 40 μL of formalin (2% in saline) to the rat's hind paw (“Formalin + morphine” group). Spontaneous pain behavior was video-recorded for 60 minutes and analyzed post hoc. Time spent licking and biting was calculated in 5-minute intervals, starting immediately after injection. The total time spent licking and biting in 60 minutes was also analyzed.
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