AV- and PI staining were performed in parallel for the detection of early apoptotic and late apoptotic/necrotic cells, respectively. 250,000 cells/well of the non-transfected HCT116, HCT116 cells transfected with either a negative control- or anti-p38α siRNA, as well as HCT116 cells in the absence/presence of p38α pharmacological inhibitors were harvested, washed with AV binding buffer (BD Biosciences, Heidelberg, Germany), and then resuspended in 50 μL binding buffer containing 5 μL of both FITC-conjugated AV and PI (BD Biosciences). Samples were incubated for 15 mins in the dark, after which they were resuspended in binding buffer up to 500 μL for FACS analysis, which was done using Guava easyCyte HT sampling flow cytometer. Data were analyzed by GuavaSoft 3.1.1 software.
Guava easycyte ht sampling flow cytometer
Manufactured by Merck Group
Sourced in United States
The Guava® easyCyte HT Sampling Flow Cytometer is a compact and efficient flow cytometry instrument designed for cell analysis. It utilizes a laser-based detection system to measure the physical and fluorescent characteristics of individual cells or particles passing through a fluidic system.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue, by Thermo Fisher Scientific (2 mentions)
Ax10 inverted microscope, by Zeiss (2 mentions)
Guava incyte assay software module, by Merck Group (2 mentions)
Av binding buffer, by BD (1 mentions)
Ab45932, by Abcam (1 mentions)
Ab150077, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay kit mts, by Promega (1 mentions)
10 protocols using guava easycyte ht sampling flow cytometer
1
Quantification of Apoptosis via Flow Cytometry
2
Annexin V and Propidium Iodide Staining
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200,000 cells/well were seeded in a 12-well plate and placed overnight in an incubator. Then, the compound was applied to the cells at the indicated concentrations. Cells were harvested 24 h later, and 250,000 cells from each well were collected, washed once with the annexin V binding buffer (Bioscience, Germany), re-suspended in 50 μL binding buffer containing 5 μL FITC-conjugated annexin V (AV, Bioscience) and 5 μL propidium iodide (PI, Bioscience). Samples were then incubated in the dark for 10–15 min at room temperature. Cell suspensions were diluted with 450 μL binding buffer and were analyzed immediately using Guava easyCyte HT sampling flow cytometer (Guava Technologies, Hayward, CA). FITC annexin V and PI were detected at excitation/emission wavelengths of 488/518 and 488/617 nm, respectively. Data were analyzed using the software GuavaSoft 3.1.1 (Guava Technologies).
3
Flow Cytometry for Cardiac Troponin T
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In conjunction with the cryopreservation, 1 million cells were collected and used for flow cytometry analysis. In short, cells were fixed in paraformaldehyde (HistoLab products AB), resuspended in methanol (−20°C) (cat. 311415, Sigma-Aldrich) and stained for cardiac troponin T using Anti-cardiac Troponin T antibody (ab45932, Abcam) as primary antibody, diluted 1:500, and Alexa flour 488 Fab2 goat anti rabbit IgG antibody (ab150077, Invitrogen), diluted 1:1000, as secondary antibody. The analysis was performed using a Guava® easyCyte HT Sampling Flow Cytometer (Merck Millipore).
4
Quantifying Virus Infection and Cell Viability
5
Cell Viability and Apoptosis Assay
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Cell viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay kit (MTS) purchased from Promega Corp. (Madison, WI) and used according to the manufacturer's protocol. For flow cytometry analysis of apoptosis, cells were treated as indicated and harvested. Cells (1×106 cells/ml) were resuspended in binding buffer and 0.5 ml of the suspension was transferred to a microfuge tube. After adding 5 μl Annexin V-FITC and 5 μl PI, cells were incubated at room temperature for 15 min in the dark. Apoptosis was analyzed by the Guava EasyCyte HT Sampling Flow Cytometer (Merck Millipore, Billerica, MA).
6
Viral Kinetics Measurement Protocols
7
Baculovirus Infection Kinetics in Insect Cells
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Sf9, VE‐CL02, High Five, VE‐High Five, SfSWT‐4, and VE‐SWT cells were infected with YFP‐AcMNPV at an MOI of 5 in triplicate. On days 2–5 post‐infection, photomicrographs and YFP fluorescence (300 ms exposure time) for each infection well were captured using a Zeiss AX10 inverted fluorescence microscope with a 20× objective and the AxioVision Rel. 4.6 program (Carl Zeiss Microscopy). After gently collecting the cells, viability was determined by staining cells with trypan blue (Thermo Fisher Scientific) and counting viable and non‐viable cells using an improved Neubauer hemocytometer under magnification of a Zeiss AX10 inverted microscope (Carl Zeiss Microscopy). To quantify YFP fluorescence, insect cells were first counted in triplicate utilizing a Guava® easyCyte HT Sampling Flow Cytometer and the guava InCyte Assay software module (EMD Millipore, Hayward, CA). YFP fluorescence was then measured using a 405‐nm laser at a green spectral imaging band (525/30 nm) of the Guava® easyCyte™ HT Sampling Flow Cytometer. The total fluorescence of each infection was determined with the Guava® InCyte Assay software module.
8
Quantifying Cell Death by PI Exclusion
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Cell death was determined using PI exclusion assay. In brief, after treatment, the cells were digested using trypsin without EDTA. Then the cells were repeatedly washed with cold PBS for three times and centrifuged at 1000 rpm for 5 min. Finally, cells were re-suspended and probed by 5 μg/mL PI for 5 min at 4 °C in the dark. Subsequently, the cells undergoing death were immediately analyzed by Guava EasyCyte HT Sampling Flow Cytometer (Merck, Millipore) with an emission wavelength at 630 nm.
9
Flow Cytometry Analysis of Cells in OCM
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Time points for flow cytometry measurements were 0, 2, 4, 6 and 10 h after transfer of cells to static liquid OCM. Samples that were incubated for 27 h in OCM were not analyzed since cell size and shape were no longer compatible with the flow cytometer. Cells were grown in OCM under the same conditions as for the RNAseq samples. At each time tested, cultures were diluted with sterile water in order to have 50–500 cells per μL in 300 μL of solution that was transferred to 96-well plates for analysis in a Guava® easyCyte HT Sampling flow cytometer (EMD Millipore Corp.). OMM that was supplemented with 1.15 g L−1 proline was used as negative control for the transition [7 (link), 8 (link)].
10
ROS Detection via H2-DCFDA Fluorescence
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H2-DCFDA is widely used for ROS detection. H2-DCFDA is a stable, nonpolar compound that readily diffuses into cells and is hydrolyzed by nonspecific esterases to DCFH. This nonfluorescent molecule is further oxidized by ROS to form the fluorescent compound DCF. The cells were incubated with 10 μM H2-DCFDA at 37°C for 30 min, then harvested and the pellets suspended in 0.5 mL of PBS. ROS generation was measured as the increase in green fluorescence intensity using the FL1 channel on a Guava EasyCyte HT Sampling Flow Cytometer (Merck Millipore, Billerica, MA).
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