Apoptotic cell death was determined using the Annexin V (conjugated to Alexa Fluor 594; excitation/emission: 590/617 nm) Apoptosis Detection Kit (BioVision, Inc., Milpitas, CA, USA) and PI. Following transfection, cells were incubated for 48 h, and then collected and stained with Annexin V/PI according to the manufacturer's protocols. The percentage of early apoptotic, late apoptotic and necrotic cells were determined using a flow cytometer (BD Bioscience) and analyzed with guavaSoft 3.1.1 software (EMD Millipore, Billerica, MA, USA). The occurrence of apoptosis in each group was investigated in ≥3 independent experiments.
Guavasoft 3
Manufactured by Merck Group
Sourced in United States, Germany
GuavaSoft 3.1.1 is a software package developed by Merck Group for the analysis and management of cellular data. The software provides tools for data acquisition, visualization, and analysis of cell-based assays. It is compatible with a range of flow cytometry and cell imaging instruments.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte 6 flow cytometer, by Merck Group (2 mentions)
Guava easycyte, by Merck Group (2 mentions)
Annexin 5, by Abcam (1 mentions)
Apoptosis detection kit, by Abcam (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Phycoerythrin pe, by Miltenyi Biotec (1 mentions)
Guava easycyte ht system, by Merck Group (1 mentions)
Guava easycyte 5, by Merck Group (1 mentions)
Facscan, by Merck Group (1 mentions)
Flow cytometry, by Merck Group (1 mentions)
Cd86 pe, by BD (1 mentions)
Cd1a apc, by BD (1 mentions)
Hla dr pe, by BD (1 mentions)
Cd14 apc, by BioLegend (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd40 fitc, by BD (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Fitc conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
5 bromo 2 deoxyuridine, by Merck Group (1 mentions)
P4170, by Merck Group (1 mentions)
13 protocols using guavasoft 3
1
Annexin V and PI Apoptosis Assay
2
Flow Cytometric Analysis of Astrocytes
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MC and SC neonatal astrocytes from WT or SOD1G93A pups were detached by trypsin-EDTA and centrifuged for 5 min at 500× g. About 5 × 105 cells were resuspended and saturated for unspecific bonds by incubating with 0.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS, pH 7.4) for 15 min at RT. Aliquots of the suspension were stained (1 h at RT) with the following fluorochrome-conjugated antibodies for flow cytometry: mouse monoclonal anti-GFAP antibody conjugated with Alexa Fluor A488 (Thermo Fisher Scientific, Cat# 53-9892-82), rat monoclonal anti-ACSA2 antibody conjugated with phycoerythrin (PE) (Miltenyi Biotec, Cat# 130-102-365) and rat monoclonal anti-TMEM119 antibody conjugated with Alexa Fluor A488 (Abcam, Cat# ab225497). For GFAP staining, cell suspensions were previously fixed and permeabilized, 20 min at 4 °C, by using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Bioscience, Cat# 554714). After staining, cells were centrifuged (5 min at 500× g) and pellets were resuspended in PBS for flow cytometry analyses. Cell debris and dead cells were excluded from analysis by 7-aminoactinomycin D (7-AAD) labeling. Data were acquired on a Guava easyCyte 6 flow cytometer (Merck Millipore, Burlington, MA, USA) and processed using the GuavaSoft 3.1.1 software (Merck Millipore).
3
Apoptosis Induction in SHG-8 Treated Cells
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To determine the stage of apoptosis induction post SHG-8 treatment, a total of 3x106 cells/well were seeded onto a 6 well plate and left to adhere overnight. Following cellular adherence, all wells were treated with control conditions and SHG-8 at 10μM and 40μM conditions for a period of 24h. Following treatment, all wells were washed with ice-cold PBS and 1x106 cells were harvested to 100μL suspensions. The SW480 cells were treated with 1μL of RNase A (10mg/mL) before being labelled with Annexin-V FITC (5μL) and propidium iodide (100μg/mL). Using the dead cell apoptosis kit with Annexin-V FITC and propidium iodide for flow cytometry (Invitrogen, Inchinnan, UK) to each 100μL cell suspension following the manufacturer’s instructions. The cell suspensions were left to incubate at RT for a period of 15 min. Following incubation, 400μL of 1x binding buffer was added to each sample and left on ice before analysing via the Guavasoft 3.1.1 software and guava easyCyte HT system (Merck Millipore, Watford, UK).
4
Flow Cytometry Analysis of Sperm Samples
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Flow cytometric analyses were performed on a Guava EasyCyte 5 cytometer (Merck KGaA, Darmstadt, Germany). The fluorescent probes used in the experiment were excited by an Argon ion 488 nm laser. Acquisitions were done using the GuavaSoft™ 3.1.1 software (Merck KGaA, Darmstadt, Germany). The non-sperm events were gated out based on scatter properties and not analyzed. A total of 40,000 events were analyzed for each sample.
5
Sperm Membrane and Organelle Evaluation
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The post-thaw semen samples were diluted with TRIS buffer (3.02% (w/v) TRIS, 1.35% (w/v) citric acid, 1.25% (w / v) fructose, in distilled water; pH 6.5, all reagents purchased from Merck, Poland) to obtain a concentration of 5 × 106 spermatozoa/mL. Each diluted sample was then divided into three aliquots of 300 μL each to assess plasma membrane integrity, acrosome integrity, and mitochondrial activity by flowcytometry Guava EasyCyte 5 (Merck KGaA, Darmstadt, Germany) cytometer. The fluorescent probes were excited by an Argon ion 488-nm laser. Detection of green fluorescence was set with an FL1 band-pass filter (525 nm / 30 nm), orange fluorescence was measured using FL2 filter (583/26 nm) and red fluorescence was measured using an FL3 filter (695/50 nm). The non-sperm events were gated out based on scatter properties and excluded from the analysis. A total of 10,000 events were analyzed per parameter for each sample. Gametes acquisitions were analyzed with the GuavaSoft™ 3.1.1 software (Merck KGaA, Darmstadt, Germany).
6
Flow Cytometry Characterization of Nanoparticles
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In the performed experiments the aqueous dispersions of P5, P5PA and P5PA-4I NPs prepared for the previous experiments were diluted 1:8 in type I deionized water 0.22 µM filtered (syringe filters, PVDF, 0.22 µm pour size, S.p.A, Milan, Italy cat#EPSPV2230) and filtered using disposable 40 µM mesh cell strainers (Euroclone S.p.A, Milan, Italy, cat#ET6040). Aliquots of the suspensions were loaded for flow cytometry analysis. Appropriate controls were run in parallel: unstained precision size standard beads 1 µm diameter (Polysciences Europe GmbH, Hirschberg an der Bergstrasse, Germany, cat# 64030-15) and deionized type I water 0.22 µM filtered. Data were acquired on a Guava easyCyte 6 flow cytometer (Merck Millipore, Burlington, MA, USA) and processed using the GuavaSoft 3.1.1 software (Merck Millipore). Table 11 collects the experimental set up and the concentration of particles (Ps) expressed as number of particles per mL.
7
Annexin V-PI Apoptosis Assay
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Analysis of apoptosis on the indicated cell lines was performed by Annexin V/propidium iodide double staining followed by Flow cytometry (Guava Technologies, Millipore, USA). The cells 2 days after transfection were collected and subjected to analysis. A minimum of 5000 cells were then analyzed by FACScan with guavaSoft 3.1.1 software (Guava Technologies, Millipore, USA) for acquisition and analysis in three independent biological replicates.
8
Phenotypic analysis of immune cells
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Cells were washed, stained with fluorophore-conjugated antibodies for 30 min at 4°C, and washed in PBS and fixed in 4% formaldehyde (Sigma-Aldrich). Samples were acquired using Guava easyCyte (Millipore), and data were analyzed using GuavaSoft 3.1.1. Antibodies were purchased from BioLegend: CD14-APC, CD40-FITC, CD80-PE, CD86-PE, CD1a-APC at 1:200 dilution, or BD Biosciences: HLA-DR-PE at 1:200 dilution. The catalogue numbers are listed in Supplementary Table S6
9
Splenocyte Isolation and Characterization
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A small portion of the spleen was used for splenocytes preparation. The splenocytes were freed of red blood cells upon treatment with lysis buffer (Solabio Co., Beijing, China), stained with CD3-APC, CD4-PerCP-Cy5.5 and CD19-PE for 30 min at 4 °C and the T and B lymphocyte subpopulation was analyzed by flow cytometry. A Guava easyCyte 6-2L system were used for the analysis with the GuavaSoft 3.1.1 software (Millipore, MA, USA), following the manufacturer's instructions.
10
Cell Cycle Analysis by BrdU-PI Staining
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The cells were treated with tetracycline (2 μg/mL) for 30 h and incubated with 20 μM BrdU (5-bromo-2-deoxyuridine; Sigma, 10280879001) for 20 min and harvested. The harvested cells were washed with the ice-cold PBS, fixed with ice-cold 70% ethanol and stored at –20 °C. The fixed cells were washed with 1% BSA/PBS, incubated in 4 N HCl with 0.5% Triton X-100 at RT for 30 min, and washed with 1% BSA/PBS three times. The cells were incubated with an anti-BrdU antibody (BD, 347580) at RT for 1 h and washed with 1% BSA/PBS three times. Then, the cells were incubated with FITC-conjugated antimouse IgG (Jackson ImmunoResearch, 115-095-003; at 1:20 dilution in 1% BSA/PBS) at RT for 30 min, washed with 1% BSA/PBS and stained DNA with propidium iodide (Sigma, P4170) (10 μg/mL) in 1% BSA/PBS at 4 °C overnight or RT for 4 h. The stained cells were applied to a Flow cytometry (Guava easyCyte: Merck). Obtained data were analyzed with an InCyte software (guava soft 3.1.1; Merck). The cell cycle gates were manually adjusted and the percentages of each cell cycle stage (G1-, S-, G2/M-phase or dead cell) were calculated.
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