Forebrain and midbrain tissues were dissected from E10.5 mouse embryos and dissociated by 0.2% Trypsin/EDTA at 37 °C for 5 min, and then filtered through a 70-μm nylon mesh filter (Corning life science, Corning, New York, USA). The isolated cells were fixed by 75% methanol. For cell cycle analysis, the cells were washed once with ice-cold PBS and labeled with propidium iodide (PI) (Sigma) at 25 °C for 1 h. Five thousand events without cell debrides were then analyzed using the Guava EasyCyte system and the InCyte software (Millipore). For analysis of Mash1-expressing cells, the cells were washed once with PBS and incubated with anti-Mash1 antibody (Abcam) with PI solution in blocking buffer (10% normal goat serum, 0.2% RNaseA, and 0.1% Triton-100X in PBS) for 1 h at 25 °C. The cells were washed and incubated with Alexa Fluor 488 anti-mouse IgG (Life Technologies) for 1 h at 25 °C. After washed with ice-cold PBS to remove the unconjugated antibodies, the cells were resuspended in PBS, and 5000 events without cell debrides were analyzed using the Guava EasyCyte system and the InCyte software (Millipore).
Incyte software
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
InCyte is a software product developed by Merck Group. It is designed to assist in the analysis and management of data acquired from laboratory equipment and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte flow cytometer, by Merck Group (34 mentions)
Guava easycyte, by Merck Group (6 mentions)
Guava flow cytometer, by Merck Group (4 mentions)
Propidium iodide, by Merck Group (3 mentions)
Guava easycyte system, by Merck Group (3 mentions)
Easycyte 8ht, by Merck Group (3 mentions)
Cell dissociation solution, by Merck Group (3 mentions)
Cd163 pe, by BD (2 mentions)
Guava easycyte 6ht 2l system, by Merck Group (2 mentions)
Guava easycyte single sample flow cytometer, by Merck Group (2 mentions)
Guava easycyte ht, by Merck Group (2 mentions)
Perm wash buffer, by BD (2 mentions)
Guava easycyte 5ht flow cytometer, by Merck Group (2 mentions)
Annexin 5 buffer, by BD (2 mentions)
Guava easycyte 5ht, by Merck Group (2 mentions)
96 well plate, by Corning (2 mentions)
Annexin 5 fitc, by Abcam (2 mentions)
Guava easy cyte 8, by Merck Group (2 mentions)
Guava easycyte 8ht flow cytometer, by Merck Group (2 mentions)
Guava easycyte 12ht benchtop flow cytometer, by Merck Group (2 mentions)
96 protocols using incyte software
1
Cell cycle and Mash1 expression analysis
2
Lipid Accumulation in Yeast Mutants
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We inoculated deletion mutants and the YKU70∆ parental strain at OD 0.1 in low nitrogen medium and cultured for 40 hr. We fixed samples by adding 180 µl cell culture to 20 µl 37% formaldehyde (Electron Microscopy Sciences, Hatfield, PA) and incubating for 15 min at room temperature. We then diluted fixed cells 1:100 in 200 µl PBS (from 10X concentrate, Thermo Fisher Scientific, 70011–44) with 0.5 M KI and 0.25 µg/mL BODIPY 493/503 (Thermo Fisher Scientific, D-3922), then incubated 30 min at room temperature. We quantified BODIPY signal for 10,000 cells per sample on a Guava HT easyCyte system (EMD Millipore) in the green channel (excitation 488 nm, emission 525 nm) using InCyte software (EMD Millipore). Due to logistical constraints, samples were processed in batches of at most 30 cultures at a time. Each batch included three biological replicates of the YKU70∆ parental strain as an internal reference. Distribution of mutant strains into these batches was not explicitly randomized, but each batch included both strains expected to accumulate more lipid and strains expected to accumulate less lipid than the parent. Each mutant was processed in at least two different batches.
3
ROS Generation Quantification in CT26 Cells
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ROS generation in vitro was investigated using dual-color flow cytometry analysis quantified after 24 h incubation of CT26 cells with each photosensitizer (20 µM). After this incubation, cells were washed with HBSS and then incubated with APF (25 µM) prepared in HBSS for the next 2 h. After this time, cells were washed twice with HBSS and irradiated with 10 J/cm2 red light (LED diode, 635 ± 20 nm, Instytut Fotonowy, Cracow, Poland). After irradiation, cells were collected by centrifugation, resuspended in 200 μL of HBSS examined using Guava® easyCyte™ flow cytometer equipped with 488 nm laser. Obtained data were analyzed using InCyte software (Merck Millipore, Burlington, MA, USA).
4
Annexin V-AF488 and Propidium Iodide Apoptosis Assay
5
BrdU Incorporation Assay for Cell Proliferation
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Cells (1–5 × 106 cells in 10 ml) were labeled with 20 µM BrdU for 20 min and collected. The cells were washed with 10 ml ice-cold PBS and fixed in 70% ethanol. After washing with 1 ml 1% BSA in PBS, the samples were incubated in 1 ml of 4 N HCl with 0.5% Triton X-100 at room temperature for 30 min and washed with 1 ml of 1% BSA in PBS three times. The cells were treated with anti-BrdU (BD) and stained with FITC-labeled anti–mouse IgG (Jackson ImmunoResearch). DNA was stained with 1 ml of 10 µg/ml propidium iodide in 1% BSA in PBS at 4°C overnight. The stained cells were applied to Guava easyCyte (Merck). Obtained data were analyzed with InCyte software (Merck).
6
Quantifying Apoptosis via Annexin V-APC
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An Annexin V-APC apoptosis detection kit (cat. no. 88–8007; eBioscience) was used for cell apoptosis analysis, according to the manufacturer's instructions. After lentiviral infection, cells were incubated for 120 h, harvested, washed with PBS buffer, and resuspended in staining buffer at a final density of ≥5×105 cells/ml. Then, 5 µl Annexin V-APC was added into 100 µl cell suspensions and incubated at room temperature for 10–15 min. Signals were detected using a Guava EasyCyte HT flow cytometer (EMD Millipore) and InCyte software (version 3.1; EMD Millipore).
7
MDM Phenotypic Characterization by Flow Cytometry
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A Guava easyCyte flow cytometer with InCyte software (EMD Millipore, USA) was used to validate both the presence of the LPS-binding-protein receptor (CD14) and the presence of α7 nAChRs. MDM cells were incubated for 1 h at 37 °C with Alexa Fluor 488 conjugated α- bungarotoxin (Life Technologies, Grand Island, NY, USA) prepared to 1 mg/mL in distilled water. Alexa Fluor 647 Anti-CD14 antibody (Imgenex, San Diego, CA), was used according to manufacturer’s directions. To determine the phenotypic characteristics of the rested MDM, cells were assayed, in triplicate, using conjugated anti-human monoclonal antibodies (mAbs) CD11b-PE-Cy7, CD11c PerCP-Cy5.5, CD80-FITC, and CD86-BB515, CD163-PE and CD68-Alexa647 (BD Biosciences, San Diego, CA). MDM were stained with mAbs for 30 minutes at 4 °C, according to the manufacturer’s protocol, and washed twice in PBS buffer prior to measuring marker expression by flow cytometry. Prior to antibody staining, MDM Fc receptors were blocked with anti-Fc antibody (BD Biosciences, San Diego, CA) according to manufacturer’s suggestion. Isotype controls (BD Biosciences, San Diego, CA) suggested by the manufacturer for each monoclonal antibody were used to calculate the change in mean fluorescence intensities.
8
Synaptosomes Oligomer Binding Assay
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Synaptosomes were treated with AβO and/or TauO for binding challenges, and the binding percentages were evaluated with flow cytometry. After assessing that the binding of AβO and/or TauO was comparable among the single human cases (Supplementary method 2), we decided to pool together an equal number of synaptosomes isolated from each subject for practical purpose. We incubated 2 million of synaptosomes for 1 h at RT without oligomers (control) as well as with AβO tagged with HyLite Fluor 647 or/and TauO tagged with Alexa Fluor™ 488 NHS Ester (Thermo Fisher Scientific) at concentrations of 0–0.5–1–2.5–5–10 μM. Synaptosomes were then pelleted, washed three times with HBK buffer, and resuspended in HBK. Oligomer fluorescence positivity was acquired by a Guava EasyCyte 8 flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore).
9
Yeast Display Screening for Fn3 Variants
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Plasmids for Fn3 variants 1.4.1 and 2.4.1, as well as wild type Fn3 (Fn3 WT), were transformed into EBY100 yeast using the Frozen-EZ Yeast Transformation Kit II (Zymo Research) following manufacturer’s protocol. Yeast were grown in SD-CAA media at 30°C and induced with SG-CAA media at 20°C with aeration. Aliquots of 106 yeast cells were simultaneously labeled with 9E10 mouse anti-c-myc antibody (1:50) and a range of concentrations of either biotinylated MSLN-Fc or biotinylated Fc fragment in a total volume of 50 μL PBSA and incubated for 45 minutes with gentle rotation at 23°C. Cells were washed with PBSA and then incubated with a goat anti-mouse PE (1:25) and streptavidin-Alexa Fluor 488 (1:700) for 20 min with gentle rotation on ice in a total volume of 25 μL PBSA, protected from light. Cells were washed with PBSA, pelleted, and resuspended in PBSA for analysis on an EMD Millipore Guava easyCyte flow cytometer. Mean fluorescence intensity for MSLN binding was determined for yeast cells displaying full length protein using InCyte software (EMD Millipore). Data was plotted and fit with a sigmoidal curve using KaleidaGraph software (Synergy). Dissociation constants (KD) were determined as the half-maximal value of the sigmoidal fit for three separate experiments for each protein variant, and the mean and standard deviation for the KD values are reported.
10
Apoptosis and Necrosis Quantification
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The cell death (apoptosis, necrosis) was analyzed by flow cytometry using Guava® easyCyte™ 6HT-2L system and InCyte Software (version 3.3, Merck KGaA, Darmstadt, Germany). The cells were seeded in the 12-well plates. The next day cells were treated with a 5 mM concentration of the CP and incubated for 24 h. Then, the cells were detached. The cell suspension was centrifuged at 300 RCF for 5 min at room temperature (RT). The pellet was resuspended in 200 µL of Annexin Binding Buffer (ABB, cat. No. V13246, Thermo Fisher Scientific, Waltham, MA, USA). The cells were stained using propidium iodide solution (cat. No. P1304MP, Thermo Fisher Scientific, Waltham, MA, USA) and Annexin V Alexa Fluor 488 (cat. No. A13201, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions (15 min, RT, dark). Next, the cell suspension was transferred to a 96-well plate (cat. No. 0030601106, Eppendorf, Hamburg, Germany) for the cytometric analysis.
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