Individual cell images were acquired using IDEAS software (Amnis Merck Millipore, Billerica, MA, USA) on a 3-laser 6-channel imaging flow cytometer (Image Stream X Mark II, Amnis Merck Millipore) with ×40 magnification. For each data file, at least 50,000 single cells were acquired—debris and doublets were excluded based on their area and aspect ratio. Single-stain controls were acquired (all channels on, no brightfield and no side scatter image), a compensation matrix was calculated and then applied to the data files using IDEAS software (Amnis Merck Millipore). Briefly, focus cells were identified using the gradient RMS feature of the brightfield channel (Ch04). Single cells were then identified from debris and cell clusters using a plot of aspect ratio vs. area of the brightfield channel. Finally, we analyzed single cells with plot based on the intensity of CMTMR and F4/80 staining (Ch03 and CH06, respectively).
Ideas software
Manufactured by Merck Group
Sourced in United States
IDEAS software is a computational platform developed by Merck Group. It is designed to assist researchers and scientists in the analysis and interpretation of data gathered through various experimental methods. The software provides tools for data visualization, statistical analysis, and model simulation, enabling users to gain insights from their research data.
Automatically generated - may contain errors
Lab products found in correlation
Imagestream x mark 2, by Merck Group (5 mentions)
Inspire software, by Merck Group (3 mentions)
Amnis imaging flow cytometer, by Merck Group (2 mentions)
Imagestream x mark 2 imaging flow cytometer, by Merck Group (2 mentions)
Amnis imagestream x mark 2, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Aria fusion sorter, by BD (1 mentions)
Rabbit anti iba1, by Novus Biologicals (1 mentions)
Anti rabbit alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Amnis flowsight imaging flow cytometer, by Merck Group (1 mentions)
Mouse anti gfap, by Cell Signaling Technology (1 mentions)
Avidin fitc, by BD (1 mentions)
7 aad, by BD (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BD (1 mentions)
Imagestreamx markii, by Merck Group (1 mentions)
Spss software v22, by IBM (1 mentions)
42 protocols using ideas software
1
Imaging Flow Cytometry Protocol for Single Cell Analysis
2
Isolation of Bead-Containing MuTuDCs
3
Imaging Flow Cytometry for Intracellular Antigen Detection
4
Multimarker Immunophenotyping of Fixed Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were gently collected and fixed for 30 min with ice-cold 4% paraformaldehyde. When necessary, cells were permeabilized with 0.1% Triton X-100 for 10 min on ice. After blocking with 3% BSA for 30 min, cells were incubated with primary antibodies overnight at 4 °C, then washed prior to incubation with secondary antibodies for 45 min at RT. Primary antibodies used were: chicken anti-integrin α-M (1:500, Aves), mouse anti-GFAP (1:500, Cell Signaling), rabbit anti-BDNF (1:500; S. Cruz), goat anti-TNFα (1:250; S. Cruz), rabbit anti-Iba1 (1:300; Novus Biologicals, NBP2-19019). Antibodies were appropriately combined in multiple labeling experiments. Secondary antibodies used were: PE-anti-chicken (1:200; S. Cruz), FITC-anti-goat (1:200; S. Cruz sc-2024), Alexa-Fluor 647-anti-rabbit (1:500; Invitrogen A31573), PE-anti-mouse (1:300; S. Cruz sc-3738). Data was acquired on the Amnis FlowSight® Imaging Flow Cytometer and analyzed with IDEAS® Software (Millipore).
5
Quantification of Tumor Cell Antigen Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies to surface and intracellular tumor cells antigens were determined. For this live and fixed/permeabilized SP2/0 Luc cells were used. SP2/0 Luc cells were fixed with 4% paraformaldehyde in PBS buffer for at least 10 min at room temperature, washed with PBS and permeabilized with 0.1% ice-cold Triton X-100 in PBS for 10 min. The cells were washed and resuspended in FACS buffer. 0.3 million SP2/0 Luc cells were used for one staining. The cells were incubated with diluted murine serum. The cells were washed with FACS buffer and incubated with antibodies to mouse immunoglobulins diluted in FACS buffer. The following antibodies to mouse immunoglobulins were used: goat a-mouse IgG FITC (Thermo Fisher Scientific, Waltham, MA, USA), anti-mouse IgM FITC (BD Pharmingen, San Jose, CA, USA), the mixture of biotin labeled antibodies to mouse IgG1, IgG2a, IgG2b, IgG3 (BD Pharmingen) with subsequent incubation with Avidin-FITC (BD Pharmingen). For elimination of dead/apoptotic cells the live SP2/0 cells were stained additionally with 7AAD (BD Pharmingen). The cells were measured with an Amnis Millipore FlowSight cytometer (Burlington, MA, USA) and analyzed with the IDEAS software (Millipore, Burlington, MA, USA). The negative control was cells stained with secondary antibodies only.
6
Multiparametric Analysis of PC12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five million PC12 cells are collected by centrifugation (500 g, 5 mn) and resuspended in 1 mL of medium containing 10 µg Hoechst 33258 (Invitrogen, Montigny le Bretonneux, France). After 30 mn incubation at 37 °C, cells were again centrifuged 500 G for 5 mn and incubated in medium containing 75 nM of Lysotracker Deep Red (Thermo Fisher Scientific, Waltham, MA, USA) and 75 µg of FDGlu (Tebu-Bio, Le Perray-en-Yvelines, France) for 1 h at room temperature. Finally, cells are centrifuged and resuspended in 20 µL of PBS + EDTA 0.5 M and analyzed using an ImageStream MKII (AMNIS) at 60X magnification at a low speed with a maximum laser power for 488 nm and 561 nm lasers (200 mW) and with 35 mW for 375 nm laser and 20 mW for 642 nm laser. Compensations were carried out using single stained controls. Around 50,000 cells were recorded in order to have sufficient a number of cells for statistical analysis. Then data were analyzed with IDEAS Software (Millipore, Billerica, MA, USA) using successive masks and features, as described in the Results section.
7
Multi-marker Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorochrome‐coupled anti‐CD8, ‐CD45, ‐CD103, ‐CD107a, ‐granzyme A, ‐IFNγ, ‐NK1.1, ‐NKp46, ‐perforin, and ‐isotype control monoclonal antibodies were purchased from eBiosciences (San Diego, CA, USA) or BD Biosciences (Franklin Lakes, NJ, USA). Uncoupled polyclonal anti‐fibronectin antibodies were purchased from AbCam. For IFNγ intracellular staining cells were stimulated with PMA/Ionomycin for four hours in the presence of GolgiPlug (BD Biosciences). All staining samples were acquired using a FACSCalibur Flow System (BD Biosciences) and data analyzed using FlowJo software (Tree Star, Ashland, OR). For imaging, cells were stained following conventional procedures and resuspended in at least 1 × 106 cells per 30 μL of staining buffer. Samples were acquired using an Amnis FlowSight instrument (Millipore, Billerica, MA, USA), and data analyzed using the IDEAS software (Millipore).
8
Cellular Localization of pSmad1/5 and INS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the cellular localization of pSmad1/5 and INS, one million of EPs was dissociated and filtered to single cell suspension as previously described and then fixed with 4% PFA with 0.1% Saponin for 30 min at 4 °C. After fixation, cells were centrifuged at 3000 × g for 3 min and washed with 0.1% Saponin, 1% BSA in PBS followed by incubation with primary and then secondary antibody diluted with 0.1% Saponin, 1% BSA/PBS. ImageStreamX MarkII (Millipore) was used to capture high-resolution single cell images to detect DAPI, INS, and pSMAD1/5 cellular localization. In total, 10,000 events were acquired, and compensation was adjusted to minimize spectral overlap between the fluorophores used in the experiment, which are DAPI, Alexa-488, and TRITC. Antibody sources, catalog numbers, and dilutions are listed in Supplementary Table 2 . Data points were analyzed by IDEAS software (Millipore).
9
Immunophenotyping of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected in ice-cold PBS and incubated with the following anti-mouse antibodies (monoclonal antibodies, mAbs): CD11c (Allophycocyanin, APC), CD80 (Fluorescein, FITC), CD86 (PE), and AGO2 (APC). All of the antibodies were purchased from Miltenyi Biotec. After antibody staining, the cells were re-suspended in PBS containing 1% paraformaldehyde and 1% FBS, and then stored at 4°C prior to flow cytometric analysis (ImageStrream mkII, Merck-Millipore, Seattle, WA, USA). Isotype-matched control mAbs (PE, FITC) were used as negative controls for all analyses. All data were quantified using the IDEAS™ Software (Millipore). All antibodies are listed in Table S3 .
10
Dietary Impact on Broiler Performance and Blood Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effect of BSFL dietary inclusion was analysed using a polynomial regression model at different degrees (1, 2 and 3) in JMP Statistics software (v14 IBM SAS Institute Inc., Cary, NC, USA) with body weight, body weight gain, ileal nutrient digestibility and also total blood count as response variables. The relationships that presented either a significant P-value or the highest R2, indicating the best relationship representation between the variables were presented in the figures with their respective best polynomial fit curves, equations and P-values for each different dietary phase (10, 21, and 42 d) as well as the entire experimental period (2 to 42 d). Blood cell count results were tested for normality using the Kolmogorov–Smirnov test (SPSS software v22, IBM, Amork, NY, USA). Total blood count results were then log-transformed to normalise data before the polynomial regression analysis was conducted P-values less than 0.05 were considered significant.
All graphs, except the graph for flow cytometry data, were built in the open source software RStudio (v1.1.453, RStudio, Inc) using the ‘ggplot2’ package (Wickham, 2016 ). The flow cytometric data were analysed performing univariate linear regression in SPSS (v22, IBM, Amork, NY, USA), and graphs were built using IDEAS software version 6.0.340.0 (Millipore,Burlington, Massachusetts , USA).
All graphs, except the graph for flow cytometry data, were built in the open source software RStudio (v1.1.453, RStudio, Inc) using the ‘ggplot2’ package (Wickham, 2016 ). The flow cytometric data were analysed performing univariate linear regression in SPSS (v22, IBM, Amork, NY, USA), and graphs were built using IDEAS software version 6.0.340.0 (Millipore,
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!