Cell toxicity was assessed with the 4-Plex Apoptosis Kit (Intellicyt, Ann Arbor, MI), which measures four parameters: cell viability (membrane integrity), caspase activity (caspase 3/7 substrate), annexin V binding (surface detection of phosphatidylserine), and mitochondrial damage (mitochondrial depolarization). One thousand cells were plated per well of a 384-well plate and were incubated with the tested compounds. Forty-eight hours later, 10 μl staining cocktail was added to the cells, and was incubated for 1 h at room temperature (RT). Data were then acquired using the iQue Screener (Intellicyt).
Ique screener
Manufactured by Intellicyt
Sourced in United States
The IQue Screener is a flow cytometry-based instrument designed for high-throughput screening applications. It provides rapid analysis and quantification of various cellular parameters, including cell count, viability, and marker expression, in a multi-well plate format.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by BD (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Thp 1 cells, by Leibniz Institute DSMZ (1 mentions)
Pe anti rabbit igg, by BioLegend (1 mentions)
Facscan, by BD (1 mentions)
Round bottom 96 well plate, by Greiner (1 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
El406 plate washer, by Agilent Technologies (1 mentions)
Freedom evo 150 liquid handling system, by Tecan (1 mentions)
Quantum alexa fluor 647 mesf beads, by Bangs Laboratories (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
High throughput flow cytometer, by Intellicyt (1 mentions)
Crl 1435, by American Type Culture Collection (1 mentions)
Canto 2 cytometer, by BD (1 mentions)
13 protocols using ique screener
1
Multiparametric Cell Toxicity Assay
2
Yeast-Based Ras-Raf Interaction Assay
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Experiments were conducted as yeast titrations (same as above). Briefly, yeast transformed with R11.1.6 were induced in SG-CAA media overnight at 20 °C. Cells were washed in stabilization buffer (same as above) supplemented with 0.1% BSA, 20 µl/L Tween-20, and 10 μM GppNHp or GDP. K-Ras G12D or WT at 10 nM was incubated with varying concentrations of the Raf RBD (residues 51–131) from 10 μM to 3.2 nM for approximately 30 minutes. Washed cells and chicken anti-cmyc antibody were added to K-Ras/Raf and incubated for approximately 3 hours, followed by incubation with goat anti-chicken-AF 488 and anti-His-AF 647 antibodies for 20 minutes. Cells were analyzed on an iQue Screener (IntelliCyt).
3
Phagocytosis Assay for Amyloid-β
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A phagocytotic assay was performed according to Ackerman et al.56 (link). In brief, THP-1 cells (DSMZ, Braunschweig, Germany) were tested for CD64 (Fcγ-RIA), CD16A (Fcγ-RIIIA) and CD32A (Fcγ-RIIA) expression. Avidin labeled fluorescent beads (FluoSpheres® NeutrAvidin®-Labeled Microspheres (1.0 μm)) were obtained from Thermo Fisher Scientific (Thermo Fisher). Biotinylated Aβ (pE3-Aβ(1–17)-PEG-Biotin) was generated at the Fraunhofer IZI (Institut fuer Zelltherapie und Immunologie, Halle, Germany). The beads were incubated with pE3-Aβ(1–17)-PEG-Biotin (500 μg/ml) over-night at 4 °C. The antigen:bead mixture was washed twice with 0.1% PBS/BSA, resuspended and transferred to a round-bottom 96 well plate at 9 × 105 beads per well. Final antibody concentrations (100 to 5 ng/ml in up to 8 replicates per concentration) were incubated for 2 hours at 37 °C. 200 μl of THP-1 cells at 1.0 × 105 cells/ml were added per well and left incubating over-night at 37 °C and 5% CO2. Cells were fixed with paraformaldehyde (4%) and analyzed with an iQue® Screener (IntelliCyt, Pasadena, USA) using the ForeCyt® 4.1 Software. After FCS/SSC gating, the fluorescent signals of whole THP-1 cells were recorded as mean fluorescent intensities (MFIs) and the cellular uptake calculated as the phagocytotic score = % of fluorescent cells x MFI of fluorescent cells.
4
Flow Cytometry Analysis of GFP Expression
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Flow cytometry analysis was performed
using an iQue Screener by Intellicyt and FCS data files were exported
and analyzed with FlowJo software. Cell populations were gated based
on forward and side scatter. Single cell populations were isolated
using forward scatter area by forward scatter height gating. Autofluorescent
cells were omitted, and remaining cells were then analyzed for GFP
levels. Percent GFP was calculated by the FlowJo software.
using an iQue Screener by Intellicyt and FCS data files were exported
and analyzed with FlowJo software. Cell populations were gated based
on forward and side scatter. Single cell populations were isolated
using forward scatter area by forward scatter height gating. Autofluorescent
cells were omitted, and remaining cells were then analyzed for GFP
levels. Percent GFP was calculated by the FlowJo software.
5
Phage Staining of Peptide-Pulsed T2 Cells
6
ADCC Assay for Cancer Cell Killing
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ADCC activity was determined using a FACS based ADCC assay. Fresh PBMCs purified from normal blood donors served as effector cells. CFSE labeled target cells (MDA-MB-231, MDA-MB-468 and MDA-MB-231, and A431) and PBMC were used at a ratio of 1:50. Indicated antibodies or fusion proteins, and target-effector cell mixture were incubated in triplicate at 37 °C for 4 h. Non-specific IgG1 and Fc-E-P125A were used as negative controls. Cells from the wells were transferred to a FACS tubes containing 0.5% BSA in PBS (pH 7.4) and propidium iodide (PI, 2μg/mL), and cell killing analyzed by flow cytometry (iQue Screener, Intellicyt or BD FACScan, BD Biosciences). ADCC was assessed by measuring frequency of dead target cells (CFSE+/PI+)/ total target cells in the sample (CFSE+/PI+ and CFSE+/PI-). Co-ordinates noted in X-axis denote antibody or fusion concentration ranging from 0.04 to 30 μg/mL while % dead cells are noted in the Y-axis. Details are in Methods
7
Evaluating CLL Cell Viability in Coculture
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This section describes the experiment shown in Figures 1 A–1C. We cultured cancer cells from four different patients with CLL (2 × 105/well) either alone or in coculture with NKTert (1 × 104/well) in 96-well glass bottom microscopy plates (zell-kontakt GmbH). Stromal cells were seeded 24 hours before the addition of leukemia cells. The samples were treated with solvent control (DMSO), 63 nM venetoclax or 10 μM fludarabine. After 72 hours the cultures were stained with the nuclear dye SiR-DNA, the viability dye Calcein and the dead cell marker PI. Images were taken with the confocal LSM710 microscope (Zeiss) equipped with climate control (37°C, 5% CO2) using a 20× objective lens. Z-stack images were acquired in triplicate wells, and within one well 4 adjacent fields were imaged.
Directly after acquisition of the confocal microscopy pictures, lymphocytes previously labeled with SiR-DNA, Calcein and PI were pipetted from each cultural condition to a 96-well round bottom plate (Greiner) for further analysis with an IQue Screener (Intellicyt). Calcein and PI signals were recorded. The following gating strategy was pursued: Exclusion of potentially remaining stromal cells, by setting of a lymphocyte gate and exclusion of doublets. The percentage of alive cells was determined by gating on Calcein positive and PI negative cells.
Directly after acquisition of the confocal microscopy pictures, lymphocytes previously labeled with SiR-DNA, Calcein and PI were pipetted from each cultural condition to a 96-well round bottom plate (Greiner) for further analysis with an IQue Screener (Intellicyt). Calcein and PI signals were recorded. The following gating strategy was pursued: Exclusion of potentially remaining stromal cells, by setting of a lymphocyte gate and exclusion of doublets. The percentage of alive cells was determined by gating on Calcein positive and PI negative cells.
8
Quantifying Cellular Uptake of siRNA
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A431-d2EGFP cells were seeded at a density of 15 000 cells/well in 96-well plates 16–20 h prior to the experiment. The p19-E18 constructs were incubated with fluorescently labeled siRNA (Seq I) at a 1:1 molar ratio for 30 min at 4°C. The p19-E18/siRNA complexes were then diluted in DMEM containing 10% FBS at varying concentrations and incubated with cells for 0–6 h in a reverse timecourse. For competition experiments, incubation was performed with 20 nM of p19-E18/siRNA complexes and varying concentrations (0–4 μM) of SUMO-E18 in complete media. After 6 h, cells were washed with PBS, trypsinized, neutralized with cold PBSA containing 2% FBS and analyzed on an iQue Screener (IntelliCyt). All liquid handling was performed using an EL406 plate washer (BioTek) and a Freedom EVO 150 liquid handling system (Tecan) to minimize variability. Background from untreated cells were subtracted from all measurements, which were then converted to number of fluorophores using Quantum Alexa Fluor 647 MESF beads following manufacturer's instructions (Bangs Laboratories).
9
Myeloid Differentiation Assay Protocol
10
GFP Analysis by Flow Cytometry
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