Neutrophil apoptosis was assessed by Annexin V staining [33 (link)]. Human neutrophils (2 × 106 cells/mL in RPMI-1640), were treated with ArtinM, IL-8, Lysis Buffer, or non-treated, and co-incubated or not with L. major during 3 and 20 h at 37°C in 5% CO2. After incubation, the cells were detached, as described above (MPO detection), washed once and suspended in 100 μL of annexin V binding buffer (140 mM NaCl, 2.5 mM CaCl2, 1.5 mM MgCl2, and 10 mM HEPES, pH 7.4), containing annexin V-FITC or PE (1 μg/mL) for 15 min. Some assays were performed on neutrophils infected with green-fluorescent L. major forms (mβT3-LV39 strain), in order to detect if the dying cells are the ones infected. Immunofluorescence staining was analyzed by flow cytometry, using a Guava EasyCyte Mini instrument (Millipore, USA).
Guava easycyte mini
Manufactured by Merck Group
Sourced in United States
The Guava EasyCyte Mini is a compact flow cytometry instrument designed for automated cell analysis. It provides a streamlined solution for cell counting, viability assessment, and basic phenotyping. The instrument is capable of detecting multiple parameters simultaneously and generating data on cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc, by Merck Group (1 mentions)
Annexin 5 fitc kit, by Thermo Fisher Scientific (1 mentions)
Cd105, by BD (1 mentions)
Cytosoft 4, by Merck Group (1 mentions)
Sodium edta, by Merck Group (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Baclight bacterial membrane potential kit, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Annexin 5 alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Annexin binding buffer, by Thermo Fisher Scientific (1 mentions)
Pi working solution, by Thermo Fisher Scientific (1 mentions)
Cytosoft software, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate, by Merck Group (1 mentions)
Fluoview fv500, by Olympus (1 mentions)
25 protocols using guava easycyte mini
1
Quantifying Neutrophil Apoptosis by Annexin V
2
Apoptosis Induction by Doxorubicin
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Induction of apoptosis was evaluated by the treatment of cells with 1 μM doxorubicin for 24 h and flow cytometry using Annexin V-FITC Kit (Invitrogen). After exposure to doxorubicin, cells were detached by trypsinization and pelleted with the supernatant. One hundred thousand cells were incubated with Annexin-V-FITC (Invitrogen) for 30 min and with propidium iodide (PI) for 30 min in Annexin-binding buffer. Green (Annexin V-FITC) and red (PI) fluorescence was accessed by counting 10,000 events under flow cytometry using Guava EasyCyte Mini (Millipore).
3
GXM Binding Assay with Jurkat Cells
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Soluble GXM was obtained from C. gattii, as reported by Wozniak and Levitz [21 (link)], at a concentration of 200 µg/mL; it was incubated with GXMR-CAR Jurkat cells (1 × 106 cells/mL) for 40 min. Cells were washed to remove unbound GXM, and murine anti-GXM monoclonal antibody (18B7 clone; Merck) was incubated with GXMR-CAR Jurkat cells for 45 min on ice. The cells were washed with cold PBS and incubated with goat anti-mouse IgG biotin-conjugated secondary antibody for 45 min. After the cells were washed, streptavidin-conjugated phycoerythrin (PE) (Thermo Fisher Scientific, Waltham, MA, USA) was added, and after 30 min, the cells were washed and analyzed using flow cytometry (Millipore Guava Easycyte Mini, Burlington, MA, USA). All the above steps were also performed with non-transduced Jurkat cells to demonstrate the absence of non-specific GXM binding. The data obtained were analyzed using the FlowJo™ software (version 10, for Windows; Ashland, OR, USA: Becton, Dickinson and Company; 2019).
4
Intracellular Transcription Factor Staining
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Samples were prepared using a Transcription Factor Staining Buffer Set (ThermoFisher/eBioscience, CA, #00–5523-00). Briefly, iPSCs were differentiated to the stage of interest. Cells were then detached using 0.25% trypsin (ThermoFisher Scientific, NY, #25200056). The cell pellet was washed twice with PBS. Cells were then fixed in fixation buffer (ThermoFisher/eBioscience) for 30 minutes at room temperature. Cells were washed once with permeabilization buffer (ThermoFisher/eBioscience) and spun at 500xg for 5 minutes. The cell pellet was resuspended in permeabilization buffer containing the primary antibody and incubated for 1 hour at room temperature. Cells were washed once with permeabilization buffer and spun at 500xg for 5 minutes. Cells pellets were resuspended in permeabilization buffer containing Alexafluor 488 secondary antibody (ThermoFisher Scientific, NY) and incubated for 30 minutes in the dark at room temperature. Cells were washed once before resuspension in permeabilization buffer and analysis using the Guava EasyCyte Mini (Millipore, MA). Data analysis was completed using FlowJo software (Version 10.5.0; FlowJo, OR) with appropriate controls. Antibodies used listed in Table S8 .
5
Intracellular Transcription Factor Staining
6
Cell Cycle Analysis by Flow Cytometry
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After a 24 h starving of FBS and hormones in the culture medium, cells were cultivated in the complete medium (FBS + TSH and other hormones) for another 24 h when cells were detached by trypsinization, pelleted with supernatant (dead cells), and fixed with 70% ethanol. After hydration in PBS, cells were treated with RNase (100 μg/ml) and DNA was stained with propidium iodide (50 μg/ml). Cell cycle was accessed by counting 10,000 events under flow cytometry using Guava EasyCyte Mini (Millipore).
7
Immunophenotyping by Flow Cytometry
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Flow cytometry was performed using Guava EasyCyte Mini (Millipore) flow cytometer. Antibodies CD105, CD73, CD90, CD34, CD45 were purchased from (BD pharmingen), isotype controls for FITC and RPE were utilized.
8
Flow Cytometric Analysis of Differentiated iPSCs
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Wild type K3 iPSCs were differentiated day 2 and day 4 of the Duncan lab differentiation protocol. Cell populations were detached using 0.25% trypsin (ThermoFisher Scientific, NY, #25200056). Cell pellets were washed twice with PBS and then incubated for 30 min at room temperature in fixation buffer from the Transcription Factor Staining Buffer Set (ThermoFisher/eBioscience, #00-5523-00). The cell pellet was washed once and then incubated in permeabilization buffer (ThermoFisher/eBioscience, #00-5523-00) with primary antibody for 1 h at room temperature. Next, samples were washed once before a 30-min incubation in permeabilization buffer containing Alexafluor 488 secondary antibody (ThermoFisher Scientific, NY). Percentage of cells positive for markers at each stage were determined by analysis using the Guava EasyCyte Mini (Millipore, MA) and FlowJo software (Version 10.5.0; FlowJo, OR).
9
Caco-2 cell dissociation and flow cytometry
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After removal of the apical and basal media, Caco–2 cells were rinsed with PBS and dissociated with trypsin/EDTA solution (Invitrogen) supplemented with 1.9 mmol/L sodium EDTA (Sigma-Aldrich) for 8 min to get a single-cell suspension. Trypsin was inhibited by adding culture media and the cells were washed with PBS. After resuspension in PBS the cells were analyzed by flow cytometry (Guava easyCyte mini, Millipore, Hayward CA, USA) and the software CytoSoft 4.2.1 (Guavatechnologies, Hayward CA, USA). The arithmetic mean of fluorescence intensity determined for 5,000 cells was used to calculate the mean fluorescence intensity (MFI) of three independent experiments. The MFI of cells incubated without PF–488 labeled peptides was used to subtract background staining.
10
Cell Cycle Analysis by Propidium Iodide Staining
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At the time of collection, the cells were harvested and washed twice with ice-cold PBS. The cells were fixed and permeabilized with 70% ice-cold ethanol at −20 °C for 4 h. The cells were washed once with PBS and resuspended in a staining solution containing propidium iodide (5 mg/mL) and RNase A (1 µg/mL). The cell suspensions were incubated for 20 min at room temperature. Then, the cell cycle profiles were measured by Guava EasyCyte Mini (Millipore, Billerica, MA, USA) detecting 10,000 cells per each group.
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