Aptamers were synthesized with 3’-end biotin modification and PAGE-grade purity at IBA GmbH or Biospring GmbH. 100 pmol of biotinylated aptamer was added to 1 × 105 cells in 100 μl PBS / BSA 1% (w/v) and incubated for 30 min at 4°C. The cells were washed with binding buffer and incubated with 100 μl PBS / BSA 1% (w/v) including streptavidin phycoerythrin (Sigma-Aldrich) (1:10) for 30 min at 4°C. The cells were washed twice and resuspended in 200 μl PBS / BSA 1% (w/v) containing propidium iodide (Invitrogen) (1:200) for exclusion of dead cells for analysis. For data acquisition and analysis the Guava easyCyte flow cytometer and the Guava Express Pro software (Millipore) were used.
Guava expresspro software
Manufactured by Merck Group
Sourced in United States
The Guava ExpressPro software is a data acquisition and analysis tool designed for flow cytometry applications. It provides a user-friendly interface for researchers to acquire, visualize, and analyze flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Streptavidin phycoerythrin, by Merck Group (1 mentions)
Guava easycyte ht facscan, by Merck Group (1 mentions)
Guava easycyte plus flow cytometry system, by Merck Group (1 mentions)
Uvb lamp, by Analytik Jena (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
Cd29 pe cy5, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Cd45 fitc, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Cd34 percp, by BD (1 mentions)
Hla abc fitc, by BD (1 mentions)
Cd31 pe, by BD (1 mentions)
Cd90 r pe, by BD (1 mentions)
Hla dr r pe, by BD (1 mentions)
Guava easycyte ht flow cytometer, by Merck Group (1 mentions)
Celastrol, by Merck Group (1 mentions)
9 protocols using guava expresspro software
1
Aptamer Binding Assay with Flow Cytometry
2
Retroviral Transduction of Tsc2+/− Eμ-Myc Lymphomas
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Tsc2+/−Eμ-Myc lymphomas were maintained in B-cell media (45 % DMEM, 45 % IMDM, 55 μM β-mercaptoethanol and 10 % fetal bovine serum) on γ-irradiated Arf−/− MEF feeder layers. Retroviral packaging was performed using ecotropic Phoenix cells according to established protocols (http://web.stanford.edu/group/nolan/_OldWebsite/protocols/pro_helper_dep.html ). Tsc2+/−Eμ-Myc lymphomas were infected with MLS retrovirus expressing shFLuc.1309 as neutral control [21 (link)] or shMcl-1.1334 [22 (link)]. The amount of GFP+ cells was determined 12 h after transduction (t = 0) and again 15 h later by flow cytometry using a Guava EasyCyte HT FACScan instrument and Guava ExpressPro software (Millipore).
3
Coronarin D Modulates Mitochondrial Potential
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Briefly, U–251 cells (3 × 104 cells/well) in 12–well plates with complete medium were incubated at 37 °C with 5% CO2 for 24 h. Then cells were treated with Coronarin D (20 and 40 µM) for 6 h. Rhodamine–123 solution (1 μg/mL in medium RPMI 1640 plus 10% FBS, 1 mL/well) was added after medium remoting. After 15 min, the medium was aspirated and all cells were washed with RPMI 1640 plus 10% FBS (1 mL/well, twice), trypsinized, and collected. The cells were analyzed (5000 events/replicate) by flow cytometry. Using the Guava® Express Pro software (Austin, TX, USA) each cell population was registered and quantified at two cell subpopulations named as viable cells (Rhodamine–123(+)); altered mitochondrial membrane potential (Rhodamine–123(–)). The analyses were done in biological duplicate of one experiment. Statistical evaluation was done by one–way ANOVA followed by Tukey’s test using GraphPad Prism software.
4
Cell Cycle and Apoptosis Analysis in Hepatocytes
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For cell cycle distribution analysis, the hepatocyte lines were collected after 24 h of treatment with a G9a inhibitor UNC0638 or mock. The cells were fixed in 1 mL ethanol (70 %) at 4 °C for 30 min and were resuspended in phosphate buffered saline containing 50 μg/mL propidium iodide (PI) and incubated at room temperature for 30 min before analysis.
For apoptosis detection assay, the hepatocyte lines were collected 24 h after treatment with ultraviolet B (UVB) irradiation (300 J/m2) by a UVB lamp (UVP, Upland, CA, USA) or hydrogen peroxide (2 mM, lasting 24 h). The cells were stained with 5 μL of FITC Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and PI (50 μg/mL) for 15 min in the dark. PI negative and Annexin V positive cells were considered early apoptotic; PI and Annexin V positive cells were considered to be in late apoptosis.
Data analysis and acquisition were performed using the Guava® EasyCyte™ Plus Flow Cytometry System and the Guava Express Pro Software (Guava Technologies, Hayward, CA, USA).
For apoptosis detection assay, the hepatocyte lines were collected 24 h after treatment with ultraviolet B (UVB) irradiation (300 J/m2) by a UVB lamp (UVP, Upland, CA, USA) or hydrogen peroxide (2 mM, lasting 24 h). The cells were stained with 5 μL of FITC Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and PI (50 μg/mL) for 15 min in the dark. PI negative and Annexin V positive cells were considered early apoptotic; PI and Annexin V positive cells were considered to be in late apoptosis.
Data analysis and acquisition were performed using the Guava® EasyCyte™ Plus Flow Cytometry System and the Guava Express Pro Software (Guava Technologies, Hayward, CA, USA).
5
Mesenchymal Origin Verification of AD-MSCs
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In order to confirm the mesenchymal origin of the obtained AD-MSC lineages, cell membrane protein expression analysis was performed by flow cytometry, using the methods previously described by the present group.11 (link), 12 (link), 13 (link), 14 (link) To that end, the adherent cells obtained in stage five were incubated with the following conjugated antibodies: CD29-PE-Cy5, CD31-PE, CD34-PerCP, CD45-FITC, CD73-PE, CD90-R-PE, CD105-PE, HLA-ABC-FITC, HLA-DR-R-PE (Becton Dickinson), according to the manufacturer's recommendations. As a negative control, cells were incubated with PBS instead of the primary antibody. A total of 10,000 events were acquired with the Guava EasyCyte flow cytometer (Guava Technologies) and analyzed with Guava ExpressPro software (Guava Technologies).
6
Flow Cytometry Analysis of Endothelial Cells
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For flow cytometry analysis of endothelial cells (ECs), cells were washed once in PBS, and enzymatically digested with 0.05% trypsin-EDTA (5 min at 37°C) and neutralized with knockout serum. Cells were centrifuged (300 x g for 5 min at room temperature) and resuspended in staining buffer (PBS + 0.05% BSA). Single cell suspensions (<1x106 cells in 100 µL per tube) were incubated for 20 min on ice with 1µL of mouse anti-human CD31-APC (eBiosciences cat# 17-0319-42). Cells were washed with 3 mL of PBS, centrifuged (300 x g for 5 min at room temperature), and resuspended in 300 µL of staining buffer prior to acquisition. Viable cells were analyzed (1,000 events acquired for each sample) using the Guava EasyCyte HT flow cytometer and Guava ExpressPro software (Luminex) (EMD Millipore, Billerica, MA, USA) at the MSM’s RCMI core facility.
7
Apoptosis Assessment of Anti-Cancer Compounds
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Cells death was assessed using AnnexinV-FITC Kit (Miltenyi Biotec, Paris, France) according to manufacturer’s protocol. Briefly, AsPC1 and HepG2 cells were respectively incubated with the gemcitabine and doxorubicin alone, or in combination with quercetin for 24 hours. Cells were then washed with phosphate buffered saline (PBS) and stained with AnnexinV-FITC and propidium iodide (PI). The fluorescence intensity of AnnexinV-FITC stained cells at 530/540 nm and PI stained cells at 675/630 nm were analyzed by Guava EasyCyte Plus capillary flow cytometer (Merck Millipore, Darmstadt, Germany) and computed using the Guava ExpressPro software (Merck/Millipore). The apoptotic potential of the tested drugs was compared to the apoptotic potential of celastrol (Sigma-Aldrich), which was used as positive control.
8
Nanoparticle Cellular Uptake Analysis
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Cellular entry of nanoparticles was appraised by confocal microscopy and flow cytometry. A qualitative study involves the usage of the CLSM 510 Confocal Microscope (Leica, Germany). FibroGRO cells were seeded in 4 wells coverslip base chambers (Nalgene, Nunc International, Naperville, IL, USA) at 5000 cells per well, treated with NLG containing 0.0625 mM lipids and FITC-SPARC siRNA and incubated 2 h at 37 °C. Before doing the imaging, FibroGRO cells were treated with Hoechst33342 (2 µg/mL, Thermo Fisher) and washed twice by PBS. All sample images were compared with untreated cells with the same imaging setting to verify the controls showed no green FITC signals and red Rhodamine signals.
Quantitative cell entry was performed via flow cytometry (Guava® easyCyte 8HT Benchtop Flow Cytometer, Merck-Millipore). In this study, 100,000 FibroGRO cells were treated with NLG containing 0.0625 mM lipids with 0.1 mol % (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt), Avanti) and then incubated 2 h at 37 °C. After trypsinization and washing, the cells were fixed at 4% paraformaldehyde (PFA) in PBS for 20 min at RT and then washed with PBS. The fluorescence inside cells was determined via flow cytometry and data was analyzed by GuavaExpress Pro software (EMD Millipore). The treated samples were normalized by the untreated cells (FibroGRO only).
Quantitative cell entry was performed via flow cytometry (Guava® easyCyte 8HT Benchtop Flow Cytometer, Merck-Millipore). In this study, 100,000 FibroGRO cells were treated with NLG containing 0.0625 mM lipids with 0.1 mol % (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt), Avanti) and then incubated 2 h at 37 °C. After trypsinization and washing, the cells were fixed at 4% paraformaldehyde (PFA) in PBS for 20 min at RT and then washed with PBS. The fluorescence inside cells was determined via flow cytometry and data was analyzed by GuavaExpress Pro software (EMD Millipore). The treated samples were normalized by the untreated cells (FibroGRO only).
9
Apoptosis and Mitochondrial Dysfunction Analysis
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HCT116 and LoVo cells at 3×105 cells/well were incubated in 6-well plates overnight at 37°C, then treated with SM-1 or 5-FU or combinations of SM-1 and 5-FU for 72 h as aforementioned. Untreated HCT116 and LoVo cells served as the control. Cells were collected, incubated with Annexin V/propidium iodide (PI) (BioLegend, Inc., San Diego, CA, USA), and measured using a Guava EasyCyte 5HT flow cytometer (EMD Millipore, Billerica, MA, USA). The compound 5,5, 6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Beyotime Institute of Biotechnology, Haimen, China) was used to assay the change in mitochondrial membrane potential (MMP). Following treatment, cells were harvested and stained with JC-1 (0.5 µmol/l) at 37°C for 20 min. The fluorescence intensity was measured using a Guava EasyCyte 5HT flow cytometer (EMD Millipore). Guava ExpressPro software (version 5.0, EMD Millipore) was used for sample analysis.
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