Cells were cultured in 50 nM LysoTracker red for 30 min. Then, cells were trypsinized and washed with PBS. Red lysosomal fluorescence of 10,000 cells per sample was determined by flow cytometry using the Cytomic FC 500MCL (BECKMAN COULTER).
Cytomic fc 500mcl
Manufactured by Beckman Coulter
Sourced in United States
The Cytomic FC 500MCL is a flow cytometry instrument designed for clinical laboratory applications. It is capable of analyzing and enumerating cells and particles in a liquid suspension.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc pi, by MultiSciences Biotech (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Rnase a, by MultiSciences Biotech (1 mentions)
Propidium iodide, by MultiSciences Biotech (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Edu cell proliferation kit with alexa fluor 488, by Beyotime (1 mentions)
Cxp software v2, by Beckman Coulter (1 mentions)
Cell cycle and apoptosis assay kit, by Beyotime (1 mentions)
Cd44 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd24 pe, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Beyotime (1 mentions)
Propidium iodide, by Beyotime (1 mentions)
Facs software, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
7 protocols using cytomic fc 500mcl
1
Lysosomal Fluorescence Analysis
2
Cell Cycle and Apoptosis Analysis
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3 × 105 DLD1 or HCT116 cells were seeded onto 6 well plates, incubated for 24 h at 37 °C, and treated as indicated. For cell cycle analysis, cells were trypsinized, resuspended and fixed with 70% ethanol at −20 °C for at least 1 h. Before analysis, the cells were resuspended in PBS containing 10 mg/mL RNaseA (Multi sciences, China) and 50 mg/mL propidium iodide (PI; Multi sciences, China) for at least 30 min. For apoptosis analysis, the supernatant was transferred to eppendorf tubes and cells were trypsinized and then collected to supernatant and washed twice with cold PBS, followed by staining with Annexin V-FITC/PI (Multi sciences, China) for 15 min. PI- and Annexin V-stained cells were analyzed immediately on a ACEA NovoCyteTM (ACEA Biosciences, USA) or Cytomic FC 500MCL (BECKMAN COULTER, USA).
3
Cell Cycle Analysis Using Flow Cytometry
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After removed the cells with 0.25% trypsin (GIBCO), the cell suspension was rinsed by pre-cooled PBS and then gently re-suspended with 70% ethanol at 4 °C for 12 h. The fixed cells were rinsed with PBS and incubated with propidium iodide containing RNase A at 37 °C for 30 min (Beyotime, C1052). The single cells were screened before flow analysis (Cytomic FC 500 MCL, Beckman Coulter, USA). Data was obtained using CXP software v2.2 (Beckman Coulter, USA), and set the parameter to FL3-620nm BP. 10,000 events were collected per sample manually determined the percentage of G1, G2 and S phases. Data in different groups were imported in Excel software (Microsoft) and generated the graph of relative cell population.
The S phase cells were detected by EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071S). The cells on coverslip were treated with pre-warmed EdU (20 µM) for 2 h, and then fixed by 4 % paraformaldehyde in PBS for 15 min and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Each process followed by PBS washing for 3 times and the cells were incubated in click additive solution (Click Reaction Buffer, CuSO4 and Azide 488) at room temperature for 30 min. The followed steps of staining the antibodies and nucleus were same as the immunofluorescence assay.
The S phase cells were detected by EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071S). The cells on coverslip were treated with pre-warmed EdU (20 µM) for 2 h, and then fixed by 4 % paraformaldehyde in PBS for 15 min and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Each process followed by PBS washing for 3 times and the cells were incubated in click additive solution (Click Reaction Buffer, CuSO4 and Azide 488) at room temperature for 30 min. The followed steps of staining the antibodies and nucleus were same as the immunofluorescence assay.
4
Cell Cycle and Apoptosis Analysis
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The cells were
seeded at 2 × 105 per well in a 6-well dish for 16
h before the experiments were performed. pEGFP transfection and propidium
iodide (PI) staining was performed according to the protocol described
above and the experimental manual of the cell cycle and apoptosis
assay kit (Beyotime). The treated cells were digested with 0.25% trypsin
and resuspended to 2 × 107/mL with buffer, and then
loaded for flow analysis (Cytomic FC 500 MCL, Beckman Coulter, USA).
seeded at 2 × 105 per well in a 6-well dish for 16
h before the experiments were performed. pEGFP transfection and propidium
iodide (PI) staining was performed according to the protocol described
above and the experimental manual of the cell cycle and apoptosis
assay kit (Beyotime). The treated cells were digested with 0.25% trypsin
and resuspended to 2 × 107/mL with buffer, and then
loaded for flow analysis (Cytomic FC 500 MCL, Beckman Coulter, USA).
5
CD44high/CD24low Population Detection
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For the detection of the CD44high/CD24low population, cells were incubated in a volume of 100 µl with 2 µl of each monoclonal antibody: CD44-PE/Cy7 and CD24-PE (eBioscience). Cells were treated on ice for 30 min and washed twice before analysis in the cytometer (Cytomic FC 500MCL; Beckman Coulter).
6
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis, cells were collected and washed with PBS and fixed in cold 70% ethanol. The samples were washed twice with PBS and incubated with a staining solution containing 20 µg/ml propidium iodide (Beyotime Technology, Shanghai, China) and 20 μg/ml RNase A (Beyotime Technology, Shanghai, China) at 37 °C for 30 min. The samples were analyzed using a flow cytometer (Cytomic FC 500 MCL, Beckman Coulter, Brea, CA, USA).
7
Annexin V Apoptosis Detection
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The CMs were examined with a FITC Annexin V apoptosis detection kit (cat# 556547, BD Biosciences, Franklin Lakes, NJ, USA) or a PE Annexin V apoptosis detection kit (cat# 559763, BD Biosciences, USA) according to the manufacturer's protocols. Apoptosis was detected by flow cytometry (Cytomic FC 500MCL, Beckman), and the apoptosis ratio was quantified by BD FACS software. The experiments were repeated three times.
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