Cells were grown in 96-well plates (Greiner, Stonehouse, UK), stimulated as indicated before fixation and immunostaining. Image acquisition was automated using an IN Cell Analyzer (GE Healthcare, Amersham, UK) imaging platform, and quantification was performed using IN Cell Analyzer work station 3.5 software. Three technical replicates were performed within each experiment, with four fields of view per well, yielding data for around 2000 cells per condition, per experiment. Additional details can be found in ESM Methods .
In cell analyzer
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The IN Cell Analyzer is a high-content screening platform designed for cellular and subcellular analysis. It captures images of cells and provides quantitative data on various cellular parameters.
Automatically generated - may contain errors
Lab products found in correlation
Acapella script, by PerkinElmer (4 mentions)
Hoechst 33342, by Merck Group (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
In cell developer toolbox, by GE Healthcare (2 mentions)
Axioscan, by Zeiss (2 mentions)
Ultrafree cl centrifugal filter, by Merck Group (2 mentions)
Fluorescence microscopy, by Olympus (2 mentions)
Lsm 780, by Zeiss (2 mentions)
96 well plate, by Greiner (2 mentions)
Calcein am, by Santa Cruz Biotechnology (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Zen microscopic software, by Zeiss (1 mentions)
Ckx41, by Olympus (1 mentions)
Anti acetylated tubulin antibody, by Merck Group (1 mentions)
Alexa fluor 488 and 594, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by AppliChem (1 mentions)
Auxin, by Merck Group (1 mentions)
60 protocols using in cell analyzer
1
Automated High-Content Cellular Imaging
2
Quantifying DNA Damage Response
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siRNA-transfected cells were re-plated into 96-well plates and left to adhere for 4 hours at 37°C before irradiation. Plates were then fixed at different time points using 3% formaldehyde. Cells were probed for γ-H2AX (Upstate/Millipore, #05–636) antibody as described previously,21 (link) and foci were imaged and quantified using an IN Cell Analyzer (GE).
3
Live/Dead Viability Assessment of Cultured Cells
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To assess cell viability, the LIVE/DEAD test was used using an InCell Analyzer 2000 automated microscope (GE Healthcare, Chicago, IL, USA). Cells of examined cell line were seeded on the surface of tested materials at constant amount of about 12 × 104 per well in 2 mL of medium suitable for a given cell line and then cultured for 48 h under standard conditions. As a control, a well with no sample was used. Before observations under a fluorescent microscope, the medium was removed and the cells were washed with PBS solution. The samples were then incubated for 15 min at room temperature in a balanced Hank salt solution containing a mixture of fluorescent dyes: Hoechst 33342 (Molecular Probes, Eugene, OR, USA), calcein AM (Santa Cruz Biotechnology, Dallas, TX, USA), and propidium iodide (Molecular Probes).
The obtained images were analyzed using InCell Analyzer (GE Healthcare) software. All cells were divided into two subpopulations, i.e., live (stained with calcein-AM—gives green color) and dead (stained with propidium iodide—red color). The total number of cells was determined based on blue fluorescence of Hoechst 33342.
The obtained images were analyzed using InCell Analyzer (GE Healthcare) software. All cells were divided into two subpopulations, i.e., live (stained with calcein-AM—gives green color) and dead (stained with propidium iodide—red color). The total number of cells was determined based on blue fluorescence of Hoechst 33342.
4
Phagocytic Capacity of THP-1 Cells
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PMA-differentiated THP-1 cells were preincubated with 2 mg/mL of E. faecalis for 24 h or with 10 μM cytochalasin D for 1 h, followed stimulation with YG beads (10 beads/cell), fecal content, E. coli (MOI 100) or P. mirabilis (MOI 100) for 3 h. Cells were stained for visualization of tubulin (1:100 dilution of anti-α-tubulin followed by a 1:1000 dilution of Alexa Fluor-594 conjugated secondsondary antibody (Invitrogen, red), YG beads, fecal content, E. coli or P. mirabilis (CFSE, green) and nuclei (DAPI, blue). To demonstrate the phagocytic efficiency of THP-1 cells, images were obtained with a confocal laser scanning microscope (LSM780; Carl Zeiss, Jena, Germany) and processed with the ZEN microscopic software (Carl Zeiss). The phagocytic activity was quantified by calculating the percentage of cells with internalized YG beads, CFSE-labeled fecal content or bacteria, as assessed using an IN cell analyzer (GE Healthcare, Freiburg, Germany).
5
Quantification of Parasite-Induced Host Cell Detachment
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HeLa cells were seeded in a 96-well plate at high density the day before infection and were infected with ∼20,000 P. berghei parasites for 2 h. Infections were performed in triplicate. The infected cells were then detached using accutase (Innovative Cell Technology) and seeded a lower density. At 48 hpi, cells of one plate were fixed with 4%PFA/PBS for 10 min. Parasite size and number were determined by automated microscopy (IN Cell analyzer, GE lifesciences; Microscopy Imaging Center, University of Bern, Switzerland) monitoring the mCherry signal in the case of KO parasites generated with 3xHA-hdhfr-yFCU-based vectors and GFP in the case of KO parasites generated with GOMO-GFP-Cherry-FACS-based vectors. At 65 hpi the detached cells in the medium of infected cells were quantified by fluorescence microscopy (Olympus CKX41). The detached cell formation rate was calculated as the percentage of parasites at 48 hpi that form detached cells. The results were statistically evaluated using Prism (GraphPad) with a one-way analysis of variance (ANOVA) test with Dunnet’s multiple comparisons.
6
Immunofluorescence Assay for Mutant-Specific p53 Antibodies
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Example 4
To test the specificity of the mutant-specific antibodies by immunofluorescence staining, the inventors again utilized the H1299-cells overexpressing the wild-type p53 or the various p53 mutants, or tumor cells lines that express endogenous mutant p53 proteins.
Fixed, transfected cells on 96 well plates were subjected to permeabilization with 0.4% Triton X-100 for 20 minutes. After rinsing with PBS, cells were blocked with 5% BSA in PBSTritonX (PBSTX) for 20 min, followed by incubation in hybridoma supernatant at 4° C. overnight. IgGs were detected using 1:1000 goat Alexaflor 488 Donkey anti-mouse IgG conjugated (Life technologies) in PBSTX with 1% BSA. Then, cells were counterstained with DAPI, and viewed with Incell Analyzer (GE Healthcare).
A distinct nuclear staining pattern was observed with the mutation-specific antibodies, which detected only their respective mutant proteins when overexpressed (
As observed with analysis by immunoblot and immunoprecipitation, the antibody against the R248Q mutant was extremely specific, and was unable to detect the related R248W mutant protein (
7
Assessing AAV9-GFP transduction in cell lines
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HEK293T, Hela or U87 cell lines grown to 60% confluency in a 96-well plate (Greiner) were pretreated with 20 µg/ml (in GM) of mouse anti-human integrin αVβ6 antibody for 2 hours at 37 °C before 50,000 vg/cell of AAV9-GFP diluted in an equal volume of GM was added and incubated at 37 °C for 24 hours. Cells were rinsed once with PBS and fixed in ice-cold 4% paraformaldehyde for 8 minutes at room temperature, washed three times for 10 minutes with PBS before counterstaining with 5 µg/ml of Hoecsht 33342 (Sigma Aldrich) at 5 µg/ml for 15 minutes. The nuclei and the GFP+ cells were imaged in the 96-well plate with an IN Cell Analyzer (GE Healthcare Life Sciences) with 10 random fields chosen per well, and the average taken from two wells. Three to four biological replicates were performed per cell line.
8
Quantifying CRISPR-Mediated Gene Editing Efficiency
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20,000 cells were infected at an MOI of ~ 1.2 to ensure 100% transduction efficiency with either virus particles of NLS-mCherry LacZ-sgRNA or NLS-GFP GOI-sgRNA. 96 h following transduction, mCherry- and GFP-expressing cells were mixed 1:1 (2,500 cells + 2,500 cells) and plated with or without olaparib (16 nM) or etoposide (100 nM) in 12-well format. During the course of the experiment, cells were subcultured when near-confluency was reached. Olaparib- or etoposide-containing medium was replaced every three days. Cells were imaged for GFP- and mCherry signal the day of initial plating (t=0) and on days 3, 6, 9, 12, 15 and 18 (olaparib), or, in a separate set of experiments, on days 5, 10, 15 and 20 (etoposide). Cells were imaged using the automatic InCell Analyzer (GE Healthcare Life Sciences, Marlborough, MA, USA) with a 4x objective. Segmentation and counting of the number of GFP-positive and mCherry-positive cells was performed using an Acapella script (PerkinElmer, Waltham, MA, USA). Efficiency of indel formation was analysed by performing PCR amplification of the region surrounding the sgRNA sequence and TIDE analysis on DNA isolated from GFP-expressing cells 9 d post-transduction.
9
Cilia Staining of Chemotherapeutic-Treated Cells
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To stain primary cilia, chemotherapeutics-treated and untreated cells were chemically fixed with 4% Formaldehyde (Merck), permeabilized with 0.1% Triton X100 (Merck) in DPBS, blocked with 1% BSA (Applichem) in DPBS, and incubated with 1:1000 dilution of anti-acetylated tubulin antibody (Sigma) or 1:500 dilution of anti-IFT88 antibody (Cat. No. 13967-1-AP, Proteintech) for 1 hour followed by incubation with 1:1000 dilution of a fluorescent secondary antibody (Life Technologies, AlexaFluor 488 and 594) for 1 hour. Nuclei were stained with Hoechst-33258 (Calbiochem). Images were acquired using an IN Cell Analyzer (GE Healthcare) at 20× magnification and were analyzed using IN Cell Developer software. In order to minimize the signal-to-noise ratio, 20 randomized fields per well were imaged at a single plane of focus.
10
Engineered hTERT-RPE1 BRCA1-/- 53BP1-/- p53-/- Cells
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