The in vitro number of receptors per cell for each tumor cell line was determined as described previously (6 (link)). Briefly, when 80% confluence was reached, each cell line was labeled with 4 mg/mL of EGF Biotin (Molecular Probes, Invitrogen, Camarillo, CA, USA), and secondarily labeled with a 1:25 dilution of Cy5-Streptavidin (Invitrogen). Control cells were stained only with the Cy-5 Streptavidin to account for autofluorescence and non-specific staining. Quantification was performed by comparison to Quantum Cy5 MESF beads, used as described by the manufacturer (Bangs Laboratory, Fisher, IN, USA). Flow cytometric data was acquired with a FACSCalibur analysis system equipped with a FACStation, and Cell Quest Acquisition software (Becton Dickinson, San Jose, CA, USA). The average fluorescence measured for the control cells was subtracted from the EGF stained cells and the number of receptors per cell was calculated assuming one molecule of biotin per EGF and three Cy5 fluorophores per Streptavidin molelecule, as specified by Invitrogen.
Facstation
Manufactured by BD
Sourced in United States
The BD FACStation is a compact, self-contained flow cytometry system designed for routine cell analysis. It provides automated sample processing, data acquisition, and analytical capabilities to support a wide range of applications in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Streptavidin cy5, by Thermo Fisher Scientific (1 mentions)
Facscalibur analysis system, by BD (1 mentions)
Cell quest acquisition software, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Citric acid, by Fujifilm (1 mentions)
Triton x 100, by Fujifilm (1 mentions)
Sodium chloride (nacl), by Nacalai Tesque (1 mentions)
Cellquest, by BD (1 mentions)
Na2hpo4, by Nacalai Tesque (1 mentions)
Pipes, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Apc annexin 5 kit, by BD (1 mentions)
Facstation, by Beckman Coulter (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Propidium iodide, by BD (1 mentions)
Wst 8 assay, by Fujifilm (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Annexin 5 fitc, by Beyotime (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
10 protocols using facstation
1
Quantification of EGFR Expression
2
Annexin V-FITC and PI Apoptosis Assay
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Cell apoptosis was detected with the FITC-Annexin V/PI staining kit. After treatment, the cells were harvested, washed in ice-cold PBS, incubated for 15 min with fluorescein-conjugated Annexin V and PI, and analyzed on a FACScan flow cytometer equipped with the FACStation data management system and Cell Quest software (all from Becton Dickinson, San Jose, CA, USA).
3
Cell Cycle Analysis via Flow Cytometry
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Briefly, cells were suspended in 500 μl of ice-cold Hank’s Balanced Saline Solution (HBSS; Sigma-Aldrich Japan, Tokyo, Japan), followed by fixation with 4.5 ml of 70% EtOH at −20°C (13 (link)). Fixed cells were centrifuged at 400 × g for 10 min, pellets were re-suspended in extraction buffer pH 7.8 which contained 45 mM Na2HPO4 (Nacalai Tesque Inc.), 25 mM citric acid (Wako Pure Chemical), and 0.1% Triton X-100 (Wako Pure Chemical) at 37°C for 25 min. Then, 300 μl of staining solution pH 6.8 containing 10 mM PIPES (Sigma-Aldrich Japan), 100 mM NaCl (Nacalai Tesque Inc.), 2 mM Mg2Cl (Wako Pure Chemical), 0.2% Triton X-100, 50 mg/ml PI (Sigma-Aldrich Japan), and 50 U/ml RNAse H (0.6 mg/ml, Takara Bio Co., Ltd., Otsu, Japan) were added to the cell suspension, and the fluorescence intensity was evaluated and analyzed in triplicate by the FACStation® and CellQuest® software (Becton-Dickinson, Franklin Lakes, NJ, USA).
4
Annexin V-FITC/PI Apoptosis Assay in CCA Cells
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The apoptosis of CCA cells in vitro was detected by Annexin V-FITC/PI staining. Both floating and adherent cells were collected after treatment (24 h) and were washed twice with ice-cold phosphate-buffered saline (PBS). The cells were then resuspended in 500 μL of binding buffer and stained with Annexin V-FITC and PI according to the manufacturer’s instructions using the Annexin V-FITC/PI Apoptosis Detection Kit. The signal was detected using a FACScan (FACStation; BD Biosciences, San Jose, CA, USA) with BD FACSCalibur and CellQuest software (using a Macintosh computer; Apple Computer, Cupertino, CA, USA). Apoptosis in the CCA cells in vivo was detected by TUNEL analysis according to the kit manufacturer’s instructions.
5
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, 2 × 106 cells were harvested, fixed with 4 mL of cold 75% ethanol at −20°C overnight, and washed twice with PBS. The cells were then resuspended in 500 μL of PBS and simultaneously stained with 200 μL of propidium iodide (50 μL/mL; Sigma-Aldrich) and incubated with 20 μL of RNase (1 mg/mL; Sigma-Aldrich) in a 37°C water bath for 15–20 min. The percentage of cells in each phase of the cell cycle was determined using a FACStation (FV500, Beckman Coulter, Brea, CA, USA) and analyzed using the Kaluza® Flow Analysis Software. Each experiment was repeated in triplicate.
For apoptosis analyses, 1 × 105 cells were stained with Annexin V and propidium iodide using an Annexin V-APC kit (BD Pharmingen™ catalog number 550474, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with a FACStation equipped with CellQuest software. Each experiment was repeated in triplicate.
For apoptosis analyses, 1 × 105 cells were stained with Annexin V and propidium iodide using an Annexin V-APC kit (BD Pharmingen™ catalog number 550474, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with a FACStation equipped with CellQuest software. Each experiment was repeated in triplicate.
6
Cell Cycle Analysis by Flow Cytometry
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Cell cycle status were detected by flow cytometry using protocols described previously [53 (link)]. Cancer cells were seeded at equal densities and maintained in culture for 24 h. Briefly, 1 × 106 cells of SKA and SKCXCR2 cells were harvested and washed twice with PBS. The cells were fixed on ice-cold 75% ethanol at 4° C for a minimum of 4 h and then washed twice with PBS. The cells were then treated with RNAse (Sigma-Aldrich) in a 37° C water bath for 20 minutes, then finally stained with propidium iodide [50 mg/ml in 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100] overnight. The cells were then analyzed by flow cytometry (FACStation, BD Biosciences) and the percentage of cells in G0/G1, S and G2/M phases were quantified utilizing FlowJo software (Tree Star Inc., Ashland, OR, USA). The assay was repeated in triplicate.
7
Cell proliferation and cell cycle analysis
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Cell proliferation was analyzed following a previously described protocol (13 (link)). Briefly, A172 cells were seeded at a density of 5×103 cells/well onto 6-well plates and cultured in DMEM with 10% FBS at 37°C for 24 h. Cells were then treated with SB (0, 0.5, 1, 2 and 4 mM) for 7 days. Following 4 days treatment, a subset of A172 cells treated with 2 mM SB were washed with PBS and cultured for a further 4 days in DMEM with 10% FBS without SB. For all cells, the culture medium was replenished every 2 d and cell numbers were recorded every 24 h using a WST-8 assay (cat no. 341-07761; Wako Pure Chemical Industries, Ltd.). Cell cycle analysis was performed using a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. Briefly, A172 cells were cultured with indicated concentrations of SB, 10, 100 nM TSA or 0.1, 1 mM methotrexate for 4 d, then collected and resuspended in 300 µl of PBS. After, 700 µl of 100% ethanol was added and kept on ice for 30 min to fix the cells. The fixed cells were resuspended in PBS supplemented with 100 µg/ml of RNase A and 50 µg/ml of propidium iodide (PI) at room temperature for 1 h. The PI stained cells were analyzed according to standard FACS protocol for cell cycle analysis with BD FACStation (v6.1, BD Biosciences, Franklin Lakes, NJ, USA).
8
Exosome Immunophenotyping by Flow Cytometry
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Isolated exosome’s were labelled according to Els J van der Vlist., et al protocol. In brief, isolated exosome’s were incubated for 60 min in 10 µl of mouse anti-CD63-PE, CD-9, CD-81 antibodies. Before data acquisition, the samples were diluted with 480 µl of 0.22 µm pre-filtered PBS (final dilution 1:25). BD FACSCalibur flow cytometer was used for data acquisition and BD FACStation was used for calculation. Data were obtained using 2 individual apogee A50/Micro flow FCs equipped with 50mW 405-nm (violet), 488-nm (blue), and 638-nm (red) lasers [23] (link).
9
Apoptosis Quantification in A2780 Cells
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Following transfection A2780 cells (2×105) were plated into 6-well plates. After 48 h, cells were digested with 0.25% trypsin, centrifuged, and the supernatants discarded. The cell pellets were washed three times with cold PBS and the supernatants discarded. The cells were then incubated with 5 µl Annexin V-FITC (C1062; Beyotime Institute of Biotechnology, Haimen, China) and 5 µl propidium iodide (PI; C1062; Beyotime Institute of Biotechnology) for 15–20 min in the dark at 25°C, and then the percentage of apoptotic cells was detected by a BD FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using the BD FACStation data management system.
10
PSEP Induces Apoptosis in C4-2 Cells
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C4-2 prostate cancer cells were seeded onto cover glass for florescence imaging and into 12-well plates for flow cytometry. C4-2 cells (2 × 106) were treated with PSEP (100 nM) for different time durations. The cells were harvested and washed in cold phosphate-buffered saline (PBS). Cells were stained with Alexa Fluor 488 Annexin V- Propidium Iodide (PI) solution for 15 min at room temperature. For imaging, fluorescence microscopy (Nikon Eclipse TE2000-S) was used to capture each channel signals separately and merged with ImageJ Plugin for colocalization. For flow cytometry, cells (1 × 104/sample) were acquired by BD FACSCalibur and analyzed using BD FACStation software.
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