96‐well imaging plates (Fisher) were coated with 0.01% poly‐L‐lysine (w/v) (Sigma‐Aldrich) for 2 h and then washed with water. Cells were labeled with 2 μM Fura‐2 AM (Life Technologies) for 30 min in cell culture medium and attached for 10 min to the plates. Intracellular Ca2+ measurements were analyzed using a Flexstation 3 fluorescence plate reader (Molecular Devices) at 340 and 380 nm excitation wavelengths. Cells were stimulated with 0.3 μM ionomycin (EMD Millipore) in 1 mM Ca2+ Ringer solution (155 mM NaCl, 4.5 mM KCl, 3 mM MgCl2, 10 mM D‐glucose, 5 mM Na HEPES) to induce SOCE. Fura‐2 fluorescence emission ratios (F340/380) were collected at 510 nm every 5 s. Ca2+ signals were quantified by analyzing the peak value of F340/380 ratios using the GraphPad Prism 9.0 software.
96 well imaging plates
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The 96-well imaging plates are a type of laboratory equipment used for cell-based assays and high-throughput screening applications. These plates provide a standardized format with 96 individual wells to accommodate cell cultures, reagents, and samples for analysis. The plates are designed to be compatible with common imaging and detection instruments, enabling efficient data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
A11034, by Thermo Fisher Scientific (2 mentions)
Pathway 855, by BD (2 mentions)
A11041, by Thermo Fisher Scientific (2 mentions)
Flexstation 3, by Molecular Devices (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (1 mentions)
H21492, by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Operetta cls high content imaging system, by PerkinElmer (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bm 220, by Merck Group (1 mentions)
Bp151, by Thermo Fisher Scientific (1 mentions)
Thapsigargin tg, by Merck Group (1 mentions)
Poly l lysine w v, by Merck Group (1 mentions)
Diode laser, by Toptica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Avantor (1 mentions)
8 protocols using 96 well imaging plates
1
Calcium Imaging of Store-Operated Calcium Entry
2
Quantifying pS65-Ub Levels via Imaging
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To quantify pS65‐Ub levels using automated high‐content imaging, fibroblasts were seeded in 96‐well imaging plates (Fisher Scientific, 08772225) and allowed to attach overnight. Cells were then treated for 0, 4, 8, or 24 h with 1 μM valinomycin and fixed in 4% paraformaldehyde after one wash with PBS. Fibroblasts were immunostained with primary antibodies against pS65‐Ub (Cell Signaling Technology, 62802; 1:1250) and HSP60 (arigo Biolaboratories, ARG10757; 1:2000) followed by incubation with secondary antibody (Invitrogen, A‐11034 and A‐11041; 1:1000) and Hoechst 33342 (Invitrogen, H21492; 1:5000). Plates were imaged on a BD Pathway 855 (BD Biosciences, San Jose, CA, USA) with a 20× objective using a 2 × 2 montage (no gaps) with laser autofocus every second frame as previously described [33 (link)]. Raw images were processed using the built‐in AttoVision V1.6 software. Regions of interest were defined as nucleus and cytoplasm using the built‐in “RING‐2 outputs” segmentation for the Hoechst channel after applying a shading algorithm. Values were normalized to 0 h and 24 h valinomycin treated control cells as 0% and 100%, respectively.
3
Intracellular Calcium Signaling in T Cells
4
Quantify p-S65-Ub levels in PINK1 mutant fibroblasts
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To evaluate top clones for immunocytochemistry and quantify p-S65-Ub levels using automated high-content imaging, WT and homozygous PINK1Q456X mutant fibroblasts were seeded in 96-well imaging plates (Fisher Scientific, 08772225) and allowed to attach overnight. Cells were treated for 2 μM valinomycin (Enzo Life Science, BML-KC140–0025) or vehicle DMSO (Sigma-Aldrich, D4540) for 24 hours and then fixed in 4% paraformaldehyde (Sigma-Aldrich, 441244) after one wash with PBS. Fibroblasts were immunostained with all clones or reference antibody against p-S65-Ub (Cell Signaling Technology, 62802, E2J6T) at 1 μg/ml and with HSP60 (arigo Biolaboratories, ARG10757, 1:2,000) followed by incubation with secondary antibody (Invitrogen, A-11034 and A-11041; 1:1,000) and Hoechst 33342 (Invitrogen, H21492; 1:5,000). Plates were imaged using an Operetta CLS High Content imaging system (Perkin Elmer, Waltham, MA, USA) with a 20x water immersion objective using a 2×2 montage (no gaps) per well. Raw images were processed using the built-in Harmony analysis software to calculate the average cytoplasmic p-S65-Ub intensity per cell in each well.
5
Quantifying p-S65-Ub Levels via Automated Imaging
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To quantify p-S65-Ub levels, automated high-content imaging was employed as recently described [43 (link),63 (link)]. In brief, cells were seeded in 96-well imaging plates (Fisher Scientific, 08772225) and allowed to attach overnight. Cells were then treated for 0 or 24 h with 1 µM valinomycin. Cells were washed once in PBS (Boston BioProducts, BM-220), fixed for 10 min in 4% paraformaldehyde (Sigma-Aldrich, 441,244) and permeabilized with 1% Triton X-100 (Fisher Scientific, BP151) in PBS. Cells were stained using different p-S65-Ub Abs (see dilution details in the table) and with Hoechst 33,342 (Invitrogen, H21492l; 1:5000). Plates were imaged on a BD Pathway 855 (BD Biosciences, San Jose, CA, USA) with a 20x objective using a 2 × 2 montage (no gaps) with laser autofocus every second frame. Raw images were processed using the built-in AttoVision V1.6 software. Regions of interest were defined as nucleus and cytoplasm using the built-in “RING-2 outputs” segmentation for the Hoechst channel after applying a shading algorithm. Background signal at 0 h was subtracted for each Ab and values were normalized to the highest value.
6
Measurement of SOCE in T cells
7
Live Cell Microscopy of Insulin and Herbal Compounds
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CHO-K1 cells were grown in 96-well imaging plates (35,000 cells/well; Nunc, Roskilde, Denmark) over night. Cell culture medium was aspirated off and, after washing the cells with PBS (VWR, Vienna, Austria), replaced by HBSS (Thermo Fisher, Waltham, MA) supplemented with 0.1% BSA (Sigma-Aldrich, Schnelldorf, Germany) for 3 hours. 3T3-L1 cells were seeded in 96-well imaging plates (20,000 cells/well) and cultured for 24 hours. The culture medium was aspirated off and, after washing the cells with PBS, replaced by serum-free culture medium overnight. Cells were incubated with insulin or herbal compounds dissolved in KRPH buffer and imaged on an Olympus IX-81 inverted microscope in objective-type TIR configuration via an Olympus 60x NA = 1.49 Plan-Apochromat objective. 96-well plates were placed on an x-y-stage (CMR-STG-MHIX2-motorized table; Märzhäuser, Wetzlar, Germany). Scanning of larger areas was supported by a laser-guided automated focus-hold system (ZDC2). The 488 nm, 561 nm and 647 nm emission of diode lasers (Toptica Photonics, Munich, Germany) were used to image GFP, tdTomato and Alexa647 fluorescence, respectively. After appropriate filtering, the fluorescence signal was recorded via an Orca-R2 CCD camera (Hamamatsu Photonics, Herrsching, Germany).
8
GLUT4 and Insulin Receptor Dynamics
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CHO-K1 cells stably expressing hIR and GLUT4-myc-GFP were a kind gift from Manoj K. Bhat (National Centre for Cell Science, University of Pune, India).[28 (link)] CHO-K1 hIR/GLUT4-myc-GFP cells were maintained in Ham’s F12 culture medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, 1% G418 and 10% fetal bovine serum (FBS) (all Life Technologies, Carlsbad, CA), and grown in a humidified atmosphere at 37°C and 5% CO2. Transient expression of pLifeact-tdTomato in CHO-K1 hIR/GLUT4-myc-GFP cells was achieved by electroporation using an Amaxa/Lonza nucleofector device (Lonza, Basel, Switzerland) according to the manufacturer (Kit T). In short, 1x106 CHO-K1 hIR/GLUT4-myc-GFP cells were transfected at 50–70% confluence with 0.18 μg DNA. Transfected cells were grown in 96 well imaging plates (Nunc, Roskilde, Denmark) for 24 hours prior imaging. 3T3-L1 cells were purchased from ATCC (Manassas, USA). 3T3-L1 cells stably expressing GLUT4-GFP were obtained from Alan Saltiel (University of Michigan). Both cell lines were maintained in DMEM high glucose culture medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin and 10% newborn calf serum (NCS) (all Life Technologies, Carlsbad, USA), and grown in a humidified atmosphere at 37°C and 5% CO2.
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