WERI-Rb1 cells or Y79 cells were harvested, fixed with 75% ice-cold ethanol in PBS and kept at 4°C. Prior to analysis, cells were washed twice with PBS and then incubated for 30 min in a propidium iodide staining solution containing 0.05 mg/ml propidium iodide, 1 mM ethylene-diaminetetraacetic acid (EDTA), 0.1% Triton X-100™ and 1 mg/ml ribonuclease A (RNase A) (all from Sigma-Aldrich, St. Louis, Missouri). The staining fluorescence intensity was measured using a cytomics FC500 MCL flow cytometer (Beckman Coulter Inc, USA) and used to determine the G1/M ratio.
Cytomics fc500 mcl flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Cytomics FC500 MCL flow cytometer is a laboratory instrument designed for the analysis and enumeration of cells. It utilizes laser-based technology to detect and measure various physical and fluorescent characteristics of individual cells as they pass through a fluid stream. The core function of the FC500 MCL is to provide researchers and clinicians with accurate and reliable cell analysis data.
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Lab products found in correlation
7 protocols using cytomics fc500 mcl flow cytometer
1
Cell Cycle Analysis by Flow Cytometry
2
Evaluating Anti-HER2 Antibody Affinity
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NIH3T3 cells expressing WT/S310F-mutant HER2 were used to evaluate the anti-HER2 antibody affinity. WT and mutant HER2 cells (6×105) were harvested using 0.25% trypsin-EDTA solution and washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.4). Then, cells were incubated with pertuzumab or trastuzumab in staining medium (PBS with 1% bovine serum albumin (BSA)) for 30 min on ice. After incubation, cells were washed twice and then incubated with FITC-conjugated goat anti-human IgG (H+L) (Beyotime, China) for 30 min. After washing, cells were suspended in 500 µL PBS and examined on a Cytomics FC500 MCL flow cytometer (Beckman Coulter, Brea, CA, USA).
3
Quantifying T cells and γδ T cells in PBMC
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Quantification of T lymphocytes (CD3+) and γδ T lymphocytes within isolated PBMC was carried out by a mouse anti-dog CD3-FITC (clone CA17.2A12, IgG1, AbD Serotec, Raleigh, NC, USA) and a mouse anti-dog TCRγδ (clone CA20.8H1, IgG2a, Prof. Peter F. Moore, Leukocyte Antigen Biology Laboratory (LABL), Davis, CA, USA [40 ]; primary antibodies followed by a goat anti-mouse IgG2a-PE secondary antibody (cat. M32204; Invitrogen™- Molecular Probes® Carlsbad, CA, USA) according to previously established protocols and optimal signal-to-noise ratio concentration [41 (link), 42 (link)]. Each incubation step with the anti-CD3 fluorochrome-labelled antibody and the secondary antibody was done in the dark for 15 min. Unstained cells and cells incubated with the secondary antibody were used as negative controls (non-specific binding). All washing steps were performed with 1 ml of PBS + 1% hi FBS. Analysis was carried out using a Cytomics FC500 MCL flow cytometer and Expo32™ ADC software (Beckman Coulter, Indianapolis, IN, USA) based on lymphocyte gating (forward scatter vs. side scatter) after acquisition of at least 10,000 cell events in the gate. The results were expressed as percentage values of CD3+ cells and TCRγδ+ cells gated on lymphocytes as well as absolute values (cells/μl) based on total lymphocyte counts.
4
Peptide Loading and TCR Binding Assay
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T2 cell line is an HLA-A*0201 positive cell line that lacks transporter-associated protein (TAP), which allows for efficient loading of endogenous peptides. Hence, only empty HLA-A*0201 exists on cell surface which can carried exogenous TCR epitope peptides [16] (link). T2 cells were cultured at 5 × 105 cells/ml in serum-free IMDM with 50 µg/ml peptide (G12V, G12D or wildtype (WT)) and 10 µg/ml human beta-2 microglobulin (β2m, Sigma) overnight at 37 °C, 5% CO2, and T2 cells only incubated with β2m were set as negative control. Cells were collected and washed twice with ice-cold PBS, then resuspended with 10 µg/ml TCRm antibodies or TCRm-ADCs in 1%BSA-PBS and incubated on ice for 30 min. After that, cells were washed twice with ice-cold PBS and stained by goat anti-human IgG (H+L)-FITC antibodies (Beyotime Biotechnology) on ice for 30 min. After twice ice-cold PBS wash, mean fluorescence intensity (MFI) of stained cells was measured by a Cytomics FC500 MCL flow cytometer (Beckman Coulter).
5
Exosome-Mediated Cell Cycle Analysis
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HUVEC were grown in 24-well plates to ~60% confluence and culture medium was replaced with EBM-2 basal medium without supplements or FBS. After 12 h incubation in basal medium, a total of 3 doses of exosomes (20 μg/ml) derived from H9C2 (one dose every 8 h) were added to HUVEC. Twenty-four hours after the first dose, cells were stained with propidium iodide (PI) according to the following procedure: floating and adhered cells were collected by centrifugation; adhered cells were detached with trypsin. Cells were resuspended in PI staining solution (50 μg/ml PI, 100 μg/ml RNAase A, 0.1% Triton X-100 and 1 mg/ml sodium citrate in distilled water) and incubated overnight at 4°C in the dark. Before acquisition, the stained cell suspensions were placed in BD TrueCount tubes (BD Biosciences) containing fluorescent beads to perform the absolute cell counting. Samples were analyzed in a Cytomics FC500 MCL flow cytometer (Beckman Coulter, USA) equipped with an argon ion laser 488 nm. PI fluorescence was collected in the 625 nm channel and cell-cycle histograms were generated excluding the cellular aggregates using the peak signal of PI. Flow cytometric data were analyzed with the FlowJo software (TreeStar Inc.) and the absolute cell counting was calculated following the recommendations of the supplier.
6
Cell Cycle Analysis of A549 and HK1 Cells
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Cell-cycle analysis of A549 and HK1 cells has been performed according to a previously reported method using a fluorochrome solution containing 50 mg/mL of propidium iodide (PI), 0.1 mg/mL of ribonuclease A, 0.1% v/v Triton X-100 and 0.1% w/v sodium citrate in d-H2O [23] . A549 and HK1 cells were each seeded in six-well plates at a density of 1x10 6 cells/well and treated for 24 h with 19 at its IC50 concentrations. There were also cells treated with DMSO (control). Cells were then harvested followed by washing with ice-cold PBS twice. Later, the pelleted cells were resuspended in 0.3-0.5 ml of the fluorochrome solution and stored overnight in the dark at 4 o C. Finally, the measurements were conducted using a Beckman Coulter Cytomics FC500 MCL flow cytometer and data analysis was performed via Weasel flow cytometry analysis software.
7
Measuring DNA Damage Response by Flow Cytometry
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Cells were seeded at a density of 1-1.5x10 6 in 10 cm 2 dishes and allowed to adhere for 24 h at 37 ºC. The cells were treated with 19 for 24 h at its IC50 concentrations and then trypsinized, collected and fixed with 1% methanol-free formaldehyde in PBS. Following a 5 min incubation at room temperature, cells were permeabilised by adding 500 µl of 0.4% Triton-X-100 in PBS and mixed gently. Cells were then rinsed with PBS, centrifuged and resuspended in 200 µl of H2AX antibody (1:3333 dilution) at room temperature for 1.5 h. Secondary antibody (goat anti-mouse Alexa Fluor 488; 1:1750 dilution) was later added and cells were incubated for 1 h at room temperature in the dark. Cells were washed with PBS and then resuspended in 300 µl of 50 µg/ml propidium iodide/ 0.1 mg/ml RNAse A in PBS followed by incubation for at least 10 min at room temperature. Finally, the measurements were conducted using a Beckman Coulter Cytomics FC500 MCL flow cytometer and data analysis was performed via Weasel flow cytometry analysis software.
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