Ibrightfl1000 system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The IBrightFL1000 system is a compact and versatile fluorescence imaging platform designed for a wide range of applications in life science research. The system features high-resolution imaging capabilities, intuitive software, and a user-friendly interface. It is capable of capturing and analyzing fluorescent signals from various samples, such as cells, tissues, and gels.

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Lab products found in correlation

12 protocols using ibrightfl1000 system

Quantitative and Qualitative RNA Analysis

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Total RNA was extracted using TRIzol (Life Technologies). Reverse transcription was performed using random priming and the Maxima First Strand cDNA Synthesis Kit for qPCR, with dsDNase (Thermo Fisher Scientific), according to the manufacturer’s guidelines. qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific), 10 mM of each primer and 20 ng of cDNA, in a CFX384 thermocycler (Bio-Rad). Variations in RNA input were corrected by analyzing the expression of GAPDH (human), β-actin (mice), or rp49 (Drosophila) as a housekeeping gene. The ΔΔCt method was used for relative quantification. Primers sequences are presented in Supplemental Table 5.
Splicing assays were performed by reverse-transcription PCR (RT-PCR), using BIOTAQ DNA Polymerase (Bioline). The primers used have been described previously and were selected to give a length difference of 10%–25% between exon-inclusion and exon-exclusion products (62 (link)). PCR amplification was performed for 20–24 cycles, and PCR products were resolved on agarose gels, stained with GelRed Nucleic Acid Gel Stain (Biotium), visualized using an iBright FL1000 system (Invitrogen), and quantified using ImageJ (NIH).

García-Puga M., Saenz-Antoñanzas A., Gerenu G., Arrieta-Legorburu A., Fernández-Torrón R., Zulaica M., Saenz A., Elizazu J., Nogales-Gadea G., Gadalla S.M., Araúzo-Bravo M.J., López de Munain A, & Matheu A. (2022). Senescence plays a role in myotonic dystrophy type 1. JCI Insight, 7(19), e159357.

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Western Blot Protein Analysis Protocol

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Cell lysates were prepared either as described below for cleavage assays or surface biotinylation assays, as appropriate. Samples were mixed with loading buffer containing SDS, 5 mM β-mercaptoethanol, and 1% dithiothreitol and heated for 10 minutes at 85°C. Proteins were separated by SDS-PAGE on 10%, 12%, or 15% polyacrylamide gels as indicated in the figure legends, transferred to nitrocellulose membrane overnight (16 hours, 55 mA, 4°C), and probed with appropriate antibodies, as indicated in the figure legends. Incubations with anti-V5 or anti-β1_intra and their respective secondary antibodies were performed using a SnapID with 10- to 20-minute incubations. Anti–α-tubulin and anti-HSP90 antibodies were incubated overnight at 4°C. Secondary antibodies for anti–α-tubulin and anti-HSP90 were incubated for 1 hour at room temperature (RT). Immunoreactive bands were detected using West Femto chemiluminescent substrate (GE Health Sciences) and imaged using an iBrightFL1000 system (Invitrogen). Immunoreactive signals from cleavage assays were quantified using ImageJ (NIH) and normalized to the level of α-tubulin and then to the vehicle-treated samples.

Bouza A.A., Edokobi N., Hodges S.L., Pinsky A.M., Offord J., Piao L., Zhao Y.T., Lopatin A.N., Lopez-Santiago L.F, & Isom L.L. (2021). Sodium channel β1 subunits participate in regulated intramembrane proteolysis-excitation coupling. JCI Insight, 6(3), e141776.

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Whole Cell Lysis and Western Blotting

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Whole cell lysates were generated by lysing cells in RIPA buffer (Thermo, Cat#89900) supplemented with Halt complete protease inhibitor cocktail (Thermo, Cat# 78429) and 1% n-dodecyl b-D-maltoside (DDM) (Thermo, Cat# BN2005). After 15 min on ice, lysates were sonicated using a Qsonica rod sonicator at 25% power for 3 s. Whole cell lysates were precleared by centrifugation at 15,000 × g for 10 min at 4°C. Protein concentrations were determined using the DC protein assay kit II (BioRad, Cat# 5000112) and standardized across all samples within an experiment. Protein lysates were reduced in Laemmli buffer (BioRad, Cat# 1610747) containing b-mercaptoethanol at 37°C for 30 min, but were not boiled to prevent aggregation of hydrophobic transmembrane regions. Equal amounts of protein were separated by SDS-PAGE and transferred to 0.2 μM nitrocellulose membranes (BioRad, Cat# 1620112). After blocking the membranes in 2% BSA in Tris-buffered saline (TBS)-Tween for 1 h at room temperature, they were incubated with primary antibodies (listed in the key resources table) at 4°C overnight and visualized either with HRP-conjugated secondary antibodies for chemiluminescence, or with up to 3 simultaneous fluorescent Alexa Fluor Plus-conjugated secondary antibodies. Images were acquired using the iBrightFL1000 system (Invitrogen).

Frenster J.D., Erdjument-Bromage H., Stephan G., Ravn-Boess N., Wang S., Liu W., Bready D., Wilcox J., Kieslich B., Jankovic M., Wilde C., Horn S., Sträter N., Liebscher I., Schöneberg T., Fenyo D., Neubert T.A, & Placantonakis D.G. (2023). PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma. Cell reports, 42(7), 112679.

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Western Blot Protein Extraction and Analysis

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Cells were washed with PBS and lysed in RIPA buffer (Thermo, Cat#89900) supplemented with Halt protease inhibitor cocktail (Thermo, Cat#78429) and 1% n-dodecyl β-D-maltoside (Thermo, Cat# BN2005). Lysates were incubated for 15 min on ice, sonicated in a water-bath Bioruptor (Diagenode, Cat# UCD-300), and precleared by centrifugation at 15,000g for 10 min at 4 °C. Protein concentrations were measured using the DC protein assay kit II (BioRad, Cat# 5000112). Laemmli buffer (BioRad, Cat# 1610747), supplemented with β-mercaptoethanol, was then added and lysates were incubated at 37 °C for 30 min. Twenty microgram of protein were separated by SDS-PAGE and transferred to 0.2 μm nitrocellulose membranes (BioRad, Cat# 1620112). Membranes were blocked in 2% bovine serum albumin (BSA) in TBS-Tween for 1 h at RT and incubated with primary antibodies (listed in Table S3) at 4 °C overnight. Following incubation with Alexa Fluor or HRP-conjugated secondary antibodies (listed in Table S4) for 1 h at RT, images were acquired using the iBrightFL1000 system (Invitrogen). Signals were detected by fluorescence or chemiluminescence (Thermo Scientific, Cat# 34577), depending on the secondary antibody. Densitometric quantification of band intensities was carried out using ImageJ.

Stephan G., Frenster J.D., Liebscher I, & Placantonakis D.G. (2022). Activation of the adhesion G protein–coupled receptor GPR133 by antibodies targeting its N-terminus. The Journal of Biological Chemistry, 298(6), 101949.

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Western Blot Analysis of Protein Samples

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Cells were washed with PBS and lysed in RIPA buffer (Thermo, Cat#89900) containing Halt protease inhibitor cocktail (Thermo, Cat# 78429) and 1% n-dodecyl β-D-maltoside (DDM) (Thermo, Cat# BN2005). Lysates were sonicated in a water-bath Bioruptor (Diagenode, Cat# UCD-300) and precleared at 15,000 x g for 10 min at 4 °C. Protein concentrations were measured using the DC protein assay kit II (BioRad, Cat# 5000112). Laemmli buffer (BioRad, Cat# 1610747) containing DTT (BioRad, Cat# 1610610) was then added and samples were incubated at 37 °C for 30 min. Twenty μg of protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (BioRad, Cat# 1620112). Membranes were blocked in 2% bovine serum albumin (BSA) in TBS-Tween for 1 hour at room temperature (RT), incubated with primary antibodies (Table 4) at 4 °C overnight, washed with TBS-Tween and incubated with Alexa Fluor or HRP-conjugated secondary antibodies (Table 4) for 1 hour at RT. Images were acquired using the iBrightFL1000 system (Invitrogen). Signals were detected by fluorescence or chemiluminescence (Thermo Scientific, Cat# 34577).

Stephan G., Erdjument-Bromage H., Liu W., Frenster J.D., Ravn-Boess N., Bready D., Cai J., Fenyo D., Neubert T, & Placantonakis D.G. (2023). Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1). bioRxiv.

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Quantifying Inflammatory Factors in Fibroblast CM

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Fibroblast cultures were washed and incubated in serum-free DMEM for 48 hours to generate conditioned medium (CM), which was then collected. The cells were then counted. CM was frozen at −80°C and analyzed using the Human Inflammation Array C3 (RayBiotech), following the manufacturer’s instructions. Briefly, CM was thawed and centrifuged for 5 minutes at 13,835.25g at room temperature. Array membranes were preincubated with 2 mL of blocking solution, incubated with CM overnight at 4°C, washed 5 times, and incubated with a biotin-conjugated antibody cocktail overnight at 4°C. After 5 washes, membranes were incubated with HRP-streptavidin for 2 hours at room temperature and then visualized using chemiluminescence detection in an iBright FL1000 system (Invitrogen). Signals were analyzed and normalized using ImageJ.

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Membrane Protein Deglycosylation and Western Blotting

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Brain membrane proteins were prepared from P60 to 90 mice as described.^{41 (link)} Complete protease inhibitors (Roche Diagnostics) were added to all solutions at twice the recommended concentration to minimize protein degradation. Deglycosylation of membrane protein samples, where indicated, was performed using PNGaseF (New England BioLabs Cat. #P0704S) as previously described.^{17 (link)} For western blotting, 50–80 µg aliquots of membrane protein were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and processed for western blotting with anti-β1_intra antibody (1:1000) (Cell Signaling Technologies 13950) or anti-Na_V1.1 Na⁺ channel antibody (K74/71) (1:200) (NeuroMab 75-023) as previously described.^{22 (link)} Anti-α-tubulin (1:1000) (Cedar Lane) or anti-transferrin receptor (TfR) (1:200) (Invitrogen H68.4 13-6800) antibodies were used as loading control, as indicated. Immunoreactive bands were detected using Supersignal West Dura Extended Duration Substrate (Therma Scientific #34076) and imaged using an iBrightFL1000 system (Invitrogen). Immunoreactive signals for the deglycosylated bands were quantified using iBright analysis software (Invitrogen) and normalized to the level of α-tubulin for each sample.

Chen C., Ziobro J., Robinson-Cooper L., Hodges S.L., Chen Y., Edokobi N., Lopez-Santiago L., Habig K., Moore C., Minton J., Bramson S., Scheuing C., Daddo N., Štěrbová K., Weckhuysen S., Parent J.M, & Isom L.L. (2023). Epilepsy and sudden unexpected death in epilepsy in a mouse model of human SCN1B-linked developmental and epileptic encephalopathy. Brain Communications, 5(6), fcad283.

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Whole Cell Lysis and Western Blotting

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Karnik S.S., Ridge K.D., Bhattacharya S, & Khorana H.G. (1993). Palmitoylation of bovine opsin and its cysteine mutants in COS cells.. Proceedings of the National Academy of Sciences of the United States of America, 90(1), 40-44.

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Quantifying NF-κB Signaling Pathway

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Anti-IκBα (1:500, Cell Signaling Technology catalog 4812), anti–NF-κB p100/p52 (1:500, Cell Signaling Technology catalog 4882), and anti–β-actin (1:2000, MilliporeSigma catalog A5441) were used. Specific bands were detected using the iBright FL1000 system (Thermo Fisher Scientific) and quantified with ImageJ. Results were expressed as a percentage from controls.

Zallocchi M., Hati S., Xu Z., Hausman W., Liu H., He D.Z, & Zuo J. (2021). Characterization of quinoxaline derivatives for protection against iatrogenically induced hearing loss. JCI Insight, 6(5), e141561.

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Western Blot Analysis of Tissue Proteins

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Approximately 100 mg of liver and spleen tissue samples were homogenized in 1 mL of RIPA buffer supplemented with 10 μL of PMSF, and then cleared by centrifugation at 12,000 rpm at 4 °C for 4 min. The supernatant was collected and its protein concentration was determined using a BCA (bicinchoninic acid) protein quantification kit. The proteins were diluted to a concentration of 50 μg/mL, mixed with a sample buffer at a ratio of 4:1, heated at 100 °C for 5 min, separated by SDS-PAGE (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in TBST buffer for 1 h. After blocking, the PVDF membrane was washed with TBST and incubated with primary antibodies (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 25 °C for 2 h. The PVDF membrane was then washed with TBST, incubated with secondary antibodies at 25 °C for 1 h, and finally incubated with Supersignal West Pico PLUS. The membrane was placed in the iBright FL1000 system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for visualization [44 (link)].

Qian Y., Zhang J., Zhou X., Yi R., Mu J., Long X., Pan Y., Zhao X, & Liu W. (2018). Lactobacillus plantarum CQPC11 Isolated from Sichuan Pickled Cabbages Antagonizes d-galactose-Induced Oxidation and Aging in Mice. Molecules, 23(11), 3026.

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