Flow cytometry acquisition and compensation was performed on a LSR Fortessa (BD Biosciences, San Jose, CA, USA). Analysis of listmode data was performed using Winlist 8.0 (Verity Software House, Inc., Topsham, ME, USA). The gating strategy is shown in Figure S4 . Briefly, the viable responder lymphocytes were hierarchically gated for single, low side scatter, viable cells with absent CD45 labeling. The percentage of proliferating cells was determined based on CFSE dye dilution compared to the CFSE labeling intensity from the non-proliferating MLR negative control sample. The full list of antibodies and reagents used in assays is available in Table S1 .
Winlist 8
Manufactured by Verity Software House
Sourced in United States
Winlist 8.0 is a software product developed by Verity Software House. It is a data analysis and visualization tool primarily used for laboratory applications. The software provides features for processing and displaying data from various laboratory instruments and experiments.
Automatically generated - may contain errors
4 protocols using winlist 8
1
Flow Cytometry Acquisition and Analysis
2
Quantifying Parasite DNA Content
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To measure the parasite DNA content, a previously described protocol (64 (link)) was followed. In brief, HFF monolayers were infected with both WT and TgOTUD3A-KO parasites. At 24 h and 36 h postinfection, parasites were harvested, syringe passaged, and filtered through 10-μm filters (CellTrics; Partec, GmbH). For a negative control, host cells only were harvested and treated the same way. Parasites fixed with filtered ethyl alcohol (EtOH; overnight fixation at −20°C) were treated with RNase, stained with the DNA dye SYTOX green (Invitrogen), and analyzed with a flow cytometer (Sony SY3200) by collecting 50,000 events for each parasite line. Data were analyzed using WinList 8.0 (Verity Software House).
3
Cell Cycle Analysis of YM155 Treatment
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Cell lines were cultured in T25 flasks in amounts ensuring 70% to 90% confluency when harvested and treated with the YM155 IC50 concentrations. After 48-hour treatment, cells were prepared for flow cytometric analysis. In short, cells were harvested, counted, and fixed in ice-cold methanol. Next, cells were washed, treated with RNase, and stained for DNA using propidium iodide and stored overnight at 4°C. Next day, cells were analyzed using an LSRII flowcytomer (BD biosciences). A blue 488-nm, 20-mW laser was used for excitation. Propidium iodide fluorescence was collected using a 610/20 band pass filter. Data analysis was performed by using WinList 8 remotely connected to ModFit LT 4 (Verity Software House, Topsham, ME) [25] (link). Each data file contained at least 10,000 single cell events. A one-compartment polynomial model was used for calculating the percentage G1, S, and G2M phase of the cell cycle. This statistical model showed the best fit.
4
DNA Content Analysis of Cell Lines
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For DNA content analysis on cell lines, the Vindeløv method was used (Vindelov et al. 1983) . In short, cells were harvested by trypsin/EDTA (Corver et al. 1995) (link), counted and 2 × 10 6 cells were divided over two polystyrene tubes. Cells were pelleted and to one tube, trout red blood cells (TRBC) were added as internal reference for DNA content. Next, solutions A, B and C, containing propidium iodide (PI), were added sequentially as described by the method. Samples were stored at 4°C and analyzed next day.
For DNA content of clinical samples, our in-housedeveloped multiparameter method was used (Corver et al. 2005 ). An LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) was used for collecting FITC, APC and/or PI fluorescence using a 530/30-, 670/14-and 610/20-nm band pass filter, respectively. 488-nm and 635-nm laser lines were used for excitation. Data were analyzed using WinList 8 and ModFit 4 (Verity Software House, Topsham, ME, USA).
For DNA content of clinical samples, our in-housedeveloped multiparameter method was used (Corver et al. 2005 ). An LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) was used for collecting FITC, APC and/or PI fluorescence using a 530/30-, 670/14-and 610/20-nm band pass filter, respectively. 488-nm and 635-nm laser lines were used for excitation. Data were analyzed using WinList 8 and ModFit 4 (Verity Software House, Topsham, ME, USA).
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