For flow cytometry for EGFP expression; EGFP NSCs and uninfected NSCs were detached with accutase, fixed by suspension in 4% paraformaldehyde (P6148, Sigma-Aldrich) for 15 minutes, and then blocked and permeabilized with flow staining buffer (5% donkey serum with 1 mg/mL saponin; D9663 and 47036, Sigma-Aldrich; in PBS) for 15 minutes. Cells were incubated with goat anti-GFP (1:100; see scWestern probing, imaging, and stripping . for product details) in flow staining buffer for 1 hour; followed by incubation with Alexa Fluor 555-labeled donkey anti-goat IgG (1:100) in flow staining buffer for 1 hour. Cells were washed twice for 5 min each with staining buffer between application of primary and secondary antibodies, and finally for 5 min with staining buffer and twice for 5 min each with PBS immediately prior to performing flow analysis. Flow cytometry was performed using an EMD Millipore EasyCyte 6HT-2L.
Easycyte 6ht 2l
Manufactured by Merck Group
Sourced in United States
The EasyCyte 6HT-2L is a compact flow cytometer designed for high-throughput analysis of biological samples. It features six fluorescence channels and two light scatter channels for the detection and analysis of various cell types and parameters. The instrument utilizes a cuvette-based flow cell and automated sample handling for efficient and reproducible measurements.
Automatically generated - may contain errors
Lab products found in correlation
D9663, by Merck Group (3 mentions)
P6148, by Merck Group (3 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta laser scanning microscope, by Zeiss (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Prism 7, by GraphPad (1 mentions)
9 protocols using easycyte 6ht 2l
1
Flow Cytometry for EGFP Expression in NSCs
2
Flow Cytometry for EGFP Expression in NSCs
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For flow cytometry for EGFP expression; EGFP NSCs and uninfected NSCs were detached with accutase, fixed by suspension in 4% paraformaldehyde (P6148, Sigma-Aldrich) for 15 minutes, and then blocked and permeabilized with flow staining buffer (5% donkey serum with 1 mg/mL saponin; D9663 and 47036, Sigma-Aldrich; in PBS) for 15 minutes. Cells were incubated with goat anti-GFP (1:100; see scWestern probing, imaging, and stripping . for product details) in flow staining buffer for 1 hour; followed by incubation with Alexa Fluor 555-labeled donkey anti-goat IgG (1:100) in flow staining buffer for 1 hour. Cells were washed twice for 5 min each with staining buffer between application of primary and secondary antibodies, and finally for 5 min with staining buffer and twice for 5 min each with PBS immediately prior to performing flow analysis. Flow cytometry was performed using an EMD Millipore EasyCyte 6HT-2L.
3
EGFP Expression Analysis in NSCs
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For flow cytometry for EGFP expression; EGFP NSCs and uninfected NSCs were detached with accutase, fixed by suspension in 4% paraformaldehyde (P6148, Sigma-Aldrich) for 15 minutes, and then blocked and permeabilized with flow staining buffer (5% donkey serum with 1 mg/mL saponin; D9663 and 47036, Sigma-Aldrich; in PBS) for 15 minutes. Cells were incubated with goat anti-GFP (1:100; see scWestern probing, imaging, and stripping. for product details) in flow staining buffer for 1 hour; followed by incubation with Alexa Fluor 555-labeled donkey anti-goat IgG (1:100) in flow staining buffer for 1 hour. Cells were washed twice for 5 min each with staining buffer between application of primary and secondary antibodies, and finally for 5 min with staining buffer and twice for 5 min each with PBS immediately prior to performing flow analysis. Flow cytometry was performed using an EMD Millipore EasyCyte 6HT-2L.
4
Quantitative HeLa Cell Labeling
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HaLo-GFP-Mito HeLa cells
were provided by the Chenoweth Lab.63 (link) Cells
were cultured using DMEM + 10% FBS + 1% Pen/Strep +1 μg/mL puromycin.
For experiments, cells were seeded in a 24- or 48-well plate the day
before at 1.0 × 105 cells/well. Cells were rinsed
with PBS and then treated with peptides in acidified Opti-MEM (0.15%
6 N HCl) for 4 h. Media was aspirated and cells were washed for 30
min with phenol red-free Opti-MEM. Cells were then incubated with
5 μM HT-TAMRA (HTag-TMR, Promega) in phenol red-free Opti-MEM
for 30 min. Cells were washed for 15 min with phenol red-free DMEM
+ 10% FBS + 1% pen/strep. Cells were rinsed with PBS and then trypsinized
and transferred to microcentrifuge tubes. Cells were pelleted and
washed twice with PBS. Cell pellets were resuspended in 250 μL
of PBS, and 200 μL was transferred to 96-well plates for flow
cytometry analysis using a Guava EasyCyte 6HT-2L benchtop flow cytometer.
Data was gated for live cells and 10 000 cells were measured
per sample. Mean fluorescence intensity was calculated from raw data,
and these values were normalized using samples with no dye added (0%
red signal) and samples with dye added but no HT-molecule added in
the preincubation step (100% red signal).
were provided by the Chenoweth Lab.63 (link) Cells
were cultured using DMEM + 10% FBS + 1% Pen/Strep +1 μg/mL puromycin.
For experiments, cells were seeded in a 24- or 48-well plate the day
before at 1.0 × 105 cells/well. Cells were rinsed
with PBS and then treated with peptides in acidified Opti-MEM (0.15%
6 N HCl) for 4 h. Media was aspirated and cells were washed for 30
min with phenol red-free Opti-MEM. Cells were then incubated with
5 μM HT-TAMRA (HTag-TMR, Promega) in phenol red-free Opti-MEM
for 30 min. Cells were washed for 15 min with phenol red-free DMEM
+ 10% FBS + 1% pen/strep. Cells were rinsed with PBS and then trypsinized
and transferred to microcentrifuge tubes. Cells were pelleted and
washed twice with PBS. Cell pellets were resuspended in 250 μL
of PBS, and 200 μL was transferred to 96-well plates for flow
cytometry analysis using a Guava EasyCyte 6HT-2L benchtop flow cytometer.
Data was gated for live cells and 10 000 cells were measured
per sample. Mean fluorescence intensity was calculated from raw data,
and these values were normalized using samples with no dye added (0%
red signal) and samples with dye added but no HT-molecule added in
the preincubation step (100% red signal).
5
Cell Cycle Analysis of Adenovirus-Infected Cells
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Chang liver cells were plated in 6-well plates at a density of 2.5 × 105 cells per well and infected with AdGFP or AdGFP-HCCR 18 h later. After 48 h of incubation, the cells were washed once with PBS, pelleted by centrifugation, and resuspended in PBS at a density of 1 × 105 cells/500 μl. Each 500 μl aliquot was added to 4.5 ml of cold 70% ethanol, and the cells were fixed for 24 h at 4°C. The cells were then pelleted, resuspended in 400 μl propidium iodide (PI) staining solution (0.1% Triton X-100, 20 μg/ml PI, and 200 μg/ml RNase A), and incubated at 37°C for 30 min before analysis in a flow cytometer (easyCyte 6HT-2L; Guava Technologies) using the Cell Cycle program, according to the manufacturer's instructions.
6
Cell Cycle Analysis by Flow Cytometry
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Cell cycle distribution was determined by flow cytometry measurement of DNA content after cells were incubated with RNase A (10 mg/ml) and propidium iodide (50 mg/ml). The cellular DNA content was analyzed on a flow cytometer (Guava EasyCyte 6HT2L). The percentage of each population was measured using the InCyte software. At least 20 000 cells were analyzed for each data point.
7
Quantifying HIV-1 Infection in CD4+ T Cells
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Unstimulated CD4+ T cells were incubated with HIV for 5 h, fixed with 2% formaldehyde and incubated with mouse anti-HIV-1 p24 antibody at 4 °C overnight, then stained with donkey anti-mouse IgG antibody conjugated with Alexa Fluor647 (Life Technologies) for 2 h. The HIV-1-p24-positive cells were measured by flow cytometry (fluorescence-activated cell sorting), 50 000 cells from each condition were analyzed by EasyCyte6HT-2L (Millipore).
8
Hyaluronan Staining of Unstimulated CD4+ T Cells
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We adapted a method from Lin37 (link) to stain HA on the unstimulated CD4+ T cell surface. Unstimulated CD4+ T cells were fixed by acid–formalin–ethanol and stained with biotinylated hyaluronan-binding protein (Calbiochem, Gibbstown, NJ, USA) overnight, then stained with Alexa488-streptavidin (Life Technologies) for 2 h. HA staining was measured by two methods: laser scanning microscopy and flow cytometry (fluorescence-activated cell sorting). For laser scanning microscopy analysis, the cells were further stained, cell membranes were stained with Texas Red (Life Technologies) and nuclei stained with DAPI (4,6-diamidino-2-phenylindole; Life Technologies). LSM510 META laser scanning microscope (Carl Zeiss Microimaging Inc., Thornwood, NY, USA) was used to scan the cells with Z-stack projections. For fluorescence-activated cell sorting analysis, 50 000 HA-stained cells were analyzed by EasyCyte6HT-2L (Millipore, Billercia, MA, USA).
9
Flow Cytometry Analysis of miR-155 Regulation
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Flow data on samples were acquired using the guava EasyCyte 6HT-2L instrument (Millipore), where 100–200 x103 cells were acquired for each sample well. Single color controls were used to determine gating as well as isotype controls were used to ascertain whether there was any non-specific antibody binding. FlowJo 10.6 software (TreeStar) was used to analyze all data. In silico analysis for the minimum free energy of the binding between miR-155 and SOCS1/SHIP1 was conducted using RNA Hybrid and Bifold software. Data were combined from each independent experiment and are presented as means ± S.D. or S.E.M. GraphPad Prism 7.0 was used to compare the mean values between groups and statistical significance of differences was determined by using either one-way ANOVA or two-tailed t-test with p ≤ 0.05 considered statistically significant.
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