HEK293 cells (ATCC) were seeded in 6-well plates at a cell density of 8 × 105 cells/well and cultured for 24 h. The cells were transfected with complex of vector DNA (the FLAG-tagged N188Q ghrelin receptor (control) or the indicated FLAG-tagged ghrelin receptor mutants) and FuGENE HD transfection reagent (Promega). The next day, transfected HEK293 cells were harvested in PBS containing 1 mM EDTA, and then incubated for 30 min on ice with 2.5 μg ml−1 anti-FLAG antibody (Wako) and 5 μg ml−1 Alexa Fluor 488–conjugated anti-mouse IgG goat polyclonal antibody (Thermo Fisher Scientific) in FACS buffer (PBS, 2% FBS, 0.05% NaN3). Cell-surface expression levels were evaluated by flow cytometry on a Guava EasyCyte Plus Flow Cytometer. FLAG-positive cells were defined as cell populations with signals greater than the top 5% of MOCK cells.
Easycyte plus flow cytometer
Manufactured by Merck Group
The EasyCyte Plus flow cytometer is a compact and user-friendly instrument designed for automated analysis of cell samples. It utilizes advanced flow cytometry technology to provide accurate and efficient data collection on various cellular parameters, including size, granularity, and fluorescence intensity.
Automatically generated - may contain errors
Lab products found in correlation
Hek293 cell, by American Type Culture Collection (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Anti flag antibody, by Fujifilm (1 mentions)
Alexa fluor 488 conjugated anti mouse igg goat polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Hek293 cells, by Fujifilm (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Guava nexin assay, by Merck Group (1 mentions)
Nexin reagent, by Merck Group (1 mentions)
9 protocols using easycyte plus flow cytometer
1
Evaluating Ghrelin Receptor Expression in HEK293 Cells
2
Cell Cycle Analysis of Drug Treated Cells
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Cells were treated with an IC30 of MK-1775, an IC25 of cisplatin, or both for 24 hours. Cells were then harvested, washed with PBS and fixed in 70% ethanol. Twenty-four hours after fixing cells were washed with PBS and then resuspended in Guava Cell Cycle reagent and analyzed on a Guava EasyCyte Plus flow cytometer.
3
Optimizing Transfection Efficiency with PEI
4
Apoptosis and Necrosis Assay in BEAS 2B Cells
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Briefly, BEAS 2B cells were seeded in 6 well plate (106 cells/well). The amount of apoptotic and necrotic cell death was assessed at 1 h and 24 h post GOx-treatment using the PE Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. 104 events were sorted and quantified using the EasyCyte™ Plus Flow Cytometer (Guava). Early apoptotic (AnxV+/7-AAD−), late apoptotic (AnxV+/7-AAD+), and necrotic (AnxV−/7-AAD+) populations. Camptothecin (10 µM) was used as a positive control.
5
Flow Cytometry Analysis of Cell Populations
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The pre-culture and growth conditions were identical to those described above for the 96-well growth assays. At the indicated time points, 1–30 μL cultures were transferred from the 96-well plate to fresh medium of the same type in a new 96-well plate to obtain a concentration of 200–500 cells/μl for flow cytometry. There were at least three biological replicates for each genotype. The flow cytometry was conducted using a Guava EasyCyte Plus flow cytometer. Each experiment was independently conducted at least twice on separate days. The data were extracted from FCS 2.0 formatted files using FlowCore (Hahne et al., 2009 (link)) (RRID:SCR_002205 ). The fluorescence levels were normalized by forward scatter to control for cell size. For each genotype, histograms of normalized fluorescence levels of 6000 cells were smoothed by Kernel density estimation and plotted using the R statistical package.
6
Intracellular Pairing of ssDNA-Conjugated CDs
7
Flow Cytometric Analysis of Ionic Gold Cytotoxicity
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To further characterize the cytotoxicity of our ionic gold delivery platform, we performed flow cytometry analysis. Cells (MCF-7, 10,000 cells/well) were grown in 24-well plate for 24 h before treating with different samples prepared for this study for 4 h. At the end of incubation, cells were trypsinized and collected in 0.2% FBS containing DPBS. Samples were analyzed using a Guava EasyCyte Plus Flow cytometer. For each sample, data from 5000 single cell events were collected for 3 min, in triplicates, and forward scatter vs. side scatter information were recorded. The results were analyzed and plotted using Flowpy.
8
Apoptosis Measurement by Flow Cytometry
9
Cell Cycle Analysis by Flow Cytometry
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Evaluation of cell cycle was performed by flow cytometry. Cells were exposed to LB100 (2.5μM) for 3 hours prior to administration of 8 Gy or sham radiation. Cells were trypsinized, fixed and stained per manufacturer's instructions with Cell Cycle Reagent, and analyzed on an EasyCyte Plus flow cytometer (Guava Technologies, Hayward, CA).
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