Analysis of DNA content and cell cycle distribution was done using muse cell cycle kit by flow cytometry. For fluorescence activated cell sorting (FACS) analysis, the treated K562 cells were incubated for 24 h. After overnight incubation cells were detached and pipetted out and spun at 300 × g for 5 min and washed once with 1 × Phosphate-buffered saline (PBS). The cells were fixed with 1 ml of ice cold ethanol and incubated at -20 °C for overnight. The ethanol fixed cells were washed once with PBS followed by 200 μL of Muse cell cycle reagent. The tubes were incubated for 30 min at dark and analyzed on Muse flow cytometer (Millipore, USA)
Muse flow cytometer
Manufactured by Merck Group
Sourced in United States
The Muse Flow Cytometer is a compact and automated flow cytometry system designed for cell analysis. It utilizes core flow cytometry principles to measure various cellular parameters, including cell count, viability, and specific protein expression. The Muse Flow Cytometer provides reliable and reproducible results, making it a versatile tool for researchers and clinicians in life science applications.
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Lab products found in correlation
Muse pi3k mapk dual pathway activation kit, by Merck Group (2 mentions)
Annexin 5 pi kit, by Thermo Fisher Scientific (2 mentions)
Temozolomide, by Selleck Chemicals (1 mentions)
Ve 822, by Selleck Chemicals (1 mentions)
Azd6738, by Selleck Chemicals (1 mentions)
Azd6244, by Selleck Chemicals (1 mentions)
Celltiter blue assay, by Promega (1 mentions)
Pe conjugated goat anti mouse igm, by Jackson ImmunoResearch (1 mentions)
Modfit 2, by Verity Software House (1 mentions)
Muse nitric oxide kit, by Merck Group (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc pi kit, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Muse cell cycle reagent, by Merck Group (1 mentions)
16 protocols using muse flow cytometer
1
Flow Cytometric Analysis of Cell Cycle
2
Cell Cycle Analysis via Flow Cytometry
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Cells were dissociated and fixed with 70% Ethanol at 4_C overnight, then stained with Muse cell cycle reagent for 30 minutes at room temperature and analyzed with Muse flow cytometer (Millipore) according to the manufacturer’s instructions. Cell cycle data were analyzed with FlowJo software.
3
Cell Cycle Analysis via Flow Cytometry
4
Assessing Cell Growth and Apoptosis
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To assess effects on cell growth, the CellTiter-Blue assay was used (Promega). In brief, 1000 to 5000 cells were plated in triplicate in 96-well plates. Then, 20–30 μL of the CellTiter-Blue reagent was added per well in 96-well plates and incubated for 1–4 h at 37 °C in 5% CO2. For drug treatments, cells in 96-well plates were cultured with various drug concentrations of AZD6244, AZD6738, VE-822, or temozolomide (Selleckchem). Vehicle (Dimethyl sulfoxide)-treated cells were used as controls and the cell survival fraction was calculated as a percentage of control cells. Fluorescence (560 nmEx/590 nmEm) was measured using a TECAN plate reader. Additionally, apoptosis assays were performed using Muse Annexin V and Dead Cell reagent (MilliporeSigma), and Bromodeoxyuridine (BrdU) incorporation assays were performed as previously described [27 (link)]. Data were acquired using Muse flow cytometer (Millipore) and analyzed with FlowJo software.
5
Cell Cycle Analysis of CD4+ T Cells
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A cell cycle analysis kit and a premixed reagent (Millipore, Billerica, MA, USA) were used for nuclear DNA intercalating staining with propidium iodide (PI), which discriminates cells at different stages of the cell cycle based on differential DNA (G0/G1, S and G2/M). CD4+ T cells were cultured for 20 hr. Isolated CD4+ cells were fixed in 70% ice-cold ethanol overnight and washed twice in PBS. The cells were analyzed on a Muse flow cytometer (Millipore) fitted with a 532 nm green diode using the cell cycle assay software. The cell cycle analysis was performed according to manufacturer's instructions.
6
Assessment of MAPK/PI3K Pathway Activation
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WM266–4 cells were seeded at 250, 000 cells/well in E-MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin in 6 well plates (Greiner Bio-One CellStar cat# 655180) and allowed to adhere overnight. After incubation cells were treated with control (25 μM dabrafenib + 25 μM trametinib), and 25 μM 2155–14 and 2155–18 for 24 and 48 h. Manufacturer’s instructions for Muse™ PI3K/MAPK Dual Pathway Activation kit (EMD Millipore MCH200108) were followed. Briefly, cells were harvested, washed with PBS, and re-suspended in 1X assay buffer supplemented with fixation buffer and incubated on ice for 10 min. Cells were permeabilized with permeabilization buffer supplied with the kit and incubated with an antibody cocktail (5 μL of Anti-Akt/PKB + 5 μL of Anti-phospho-Akt). Cells were analyzed on Muse flow cytometer (EMD Millipore) using PI3K/MAPK Dual Pathway sub-routine.
7
Ceramide Levels in p53 HCT116 Cells
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Ceramide levels present in the cell membrane of p53+/+ HCT116 cells were assessed by flow cytometry. Cells were treated with unlabeled mAbs (20 μg/ml) or 125I-mAbs (4 MBq/ml) at 37°C for 1 h. After three washes, cells were fixed in 3.7% (v/v) paraformaldehyde for 15 min. Then, cells were washed three times in PBS buffer and incubated with the anti-ceramide 15B4 mAb at a dilution of 1:50 (Alexis Biochemicals) at 37°C for 1 h. After three washes in PBS—2% FCS, cells were incubated with PE-conjugated goat anti-mouse IgM at a dilution of 1:100 (Jackson ImmunoResearch) in the dark for 1 h. Cells were then washed three times and suspended in PBS for analysis using a Muse flow cytometer (Merck Millipore, Fullerton, CA). The y axis corresponds to the total number of analyzed cells (arbitrary units), while the x axis represents the fluorescence signal intensity (PE-conjugated goat mAb anti-ceramide 15B4 mAb). The numbers provided in the red boxes correspond to the proportion of ceramide-positive cells within the total population of cells compared with the nontreated control.
8
Cytotoxicity of mHSP and Grp94 Vaccines
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The cytotoxicities of the mHSP/peptide and Grp94/peptide were tested using flow cytometry. The mHSP/peptide vaccine was applied at four different concentrations to the fibroblast L929 cell line, and the same procedure was used for the Grp94/peptide vaccine. The four different concentrations of the mHSP/peptide vaccine ranged from 0 to 30 µg/mL, and the Grp94/peptide vaccine concentrations ranged from 0 to 300 ng/mL. The L929 cells were cultured in 6-well plates, and vaccines were added to 4 wells at different concentrations. The plates were placed in a carbon dioxide cell incubator for 24 h. A Muse flow cytometer (Merck Company) was used for the L929 viability tests. The flow cytometry test for L929 cell viability treated with different vaccine concentrations followed the instructions of the Muse Count/Viability manual.
9
PM2.5-Induced Cell Cycle Analysis
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Cells were treated with PM2.5 suspension for 48 h, centrifuged at 13,000 × g for 3 min at 4°C and washed twice with PBS. Cells were resuspended with PBS, and 2 volumes of 5 ml ethanol were slowly added and mixed, stationary set was at 4°C. The cells were washed twice with PBS, resuspended in 1 ml of PI solution and incubated at room temperature for 30 min. The cell cycle was detected by flow cytometry (Muse™ flow cytometer; Merck Millipore, Burlington, MA, USA) and the data were analyzed with ModFit 2.0 software (Verity Software House, Inc., Berlin, Germany).
10
Annexin V-PI Cell Apoptosis Assay
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Cells were stained using an Annexin V-PI kit (Invitrogen). A total of 1 × 105 cells were suspended in 500 µL binding buffer, and then stained with 5 µL Annexin V‐EGFP and 5 mL Propidium Iodide, respectively, at room temperature for 10 min in the dark. The cell apoptosis was analyzed on a MUSE™ flow cytometer (Merck Millipore, USA). Lower left quadrant (LL) represented viable cells; upper left quadrant (UL) represented necrotic cells; lower right quadrant (LR) represented early apoptotic cells; upper right quadrant (UR) represented late apoptotic cells. The apoptotic ratio (%) was calculated as the cell ratio in LR + UR.
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