The binding affinity of ZV05 and ZV0508 to 5T4‐positive human cancer cell lines was determined by flow cytometry. 3 × 105 cells were incubated with ZV05 or ZV0508 in PBS + 1% bovine serum albumin (w/v) for 30 minutes on ice. After incubation, cells were washed twice with PBS and then incubated with fluorescein isothiocyanate (FITC)‐labeled goat anti‐human IgG (H+L) polyclonal antibody at 1:300 for 30 minutes on ice. After washing, cells were examined on a Beckman Coulter Cytomics FC500 Flow Cytometer. Data analysis was performed using CXP analysis 2.2 (Beckman Coulter) and the geometric mean of fluorescence intensity ratio (MFI) of each cell line was determined.
Cxp analysis 2
Manufactured by Beckman Coulter
Sourced in United States
The CXP Analysis 2.0 software is a data analysis tool developed by Beckman Coulter for flow cytometry applications. It provides users with the ability to analyze and interpret data generated from Beckman Coulter's flow cytometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Fc500mcl, by Beckman Coulter (3 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (1 mentions)
Fitc labeled secondary antibody, by Beyotime (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Annexin 5 pi detection kit, by Keygen Biotech (1 mentions)
Cd90 fitc, by Thermo Fisher Scientific (1 mentions)
Cd105 fitc, by Thermo Fisher Scientific (1 mentions)
Cd73 fitc, by Thermo Fisher Scientific (1 mentions)
Cd34 fitc, by Thermo Fisher Scientific (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
8 protocols using cxp analysis 2
1
Binding Affinity of ZV05 and ZV0508 to 5T4-positive Cancer Cells
2
Quantification of Antibody Binding to CD20
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Binding of antibody or ADCs to cell-surface CD20 was assessed by flow cytometry (FC500MCL, Beckman Coulter, Brea, CA, USA) on CD20-positive Daudi cells. 1 × 106 cells were incubated with 5 μg/mL antibody or ADCs in staining medium containing PBS (pH 7.4) with 1% (w/v) bovine serum albumin (BSA) on ice for 30 min, and then washed twice with ice-cold PBS to remove unbound antibody or ADCs. Cells were stained with FITC-labeled secondary antibody (Beyotime, Shanghai, China) at a dilution of 1:250 in ice-cold staining medium, incubated 30 min on ice, and washed as above. Labeled cells were examined by flow cytometry gated to exclude nonviable cells. Data was analyzed using CXP Analysis 2.2 (Beckman Coulter, Brea, CA, USA) and the background-corrected mean fluorescence intensity was determined.
3
Apoptosis Measurement in PASMCs
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The apoptosis of PASMCs was measured using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry kit (BD Biosciences) according to the manufacturers' instructions. PASMCs were washed twice with ice-cold PBS and resuspended in 200 µl of binding buffer at a concentration of 1x106 cells/ml. Annexin V-FITC and PI (10 µl of each) were added, and the cells were incubated for 30 min at 4˚C in the dark. Finally, 300 µl of binding buffer was added and the cells were analyzed by flow cytometry (Cytomics FC 500; Beckman Coulter, Inc.) within 1 h using CXP Analysis 2.0 software (Beckman-Coulter).
4
Annexin V-FITC Apoptosis Assay
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The apoptosis assay was performed as described previously [28 (link)]. In brief, at the end of each treatment HCE cells viability was assessed by flow cytometry using an Annexin V apoptosis detection kit (MBL, Nagoya, Japan). One μg/ml of Annexin V-FITC and 1 μg/ml of Propidium iodide (PI) were added to the cell suspension, and the mixture was incubated in the dark for 5 min at room temperature. Without washing, the cells were placed in 500 μl of Annexin V binding buffer and kept on ice, and within 5 min were evaluated using a Coulter FC-500 flow cytometer (Beckman-Coulter, Fullerton, CA, USA). Flow cytometry data were analyzed using CXP Analysis 2.0 software (Beckman-Coulter, Miami, FL, USA).
5
Apoptosis Detection Using Annexin-V/PI
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Cellular apoptosis was detected using the Annexin-V/PI detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. The transfected cells were suspended and centrifuged at 1,000 × g for 5 min at room temperature. The supernatant was then removed, and the cells were resuspended in 200 µl binding buffer. The cells were stained with 10 µl Annexin V-FITC and 10 µl PI and then incubated for 30 min at 4°C in the dark. Cellular apoptosis was detected under a FC500 MCL flow cytometer (Beckman Coulter, Inc.) and analyzed using CXP Analysis 2.0 software (Beckman Coulter, Inc.). The apoptotic rate was calculated by the percentage of early and late apoptotic cells.
6
Multiparametric Flow Cytometry Characterization
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The cells (1x106 per group) were resuspended in 100 µl of flow buffer (cat. no. PAB180076; Bioswamp Wuhan Beinle Biotechnology Co., Ltd.) in an Eppendorf tube and 2 µl of CD45-FITC (cat. no. 11-9459-42, eBioscience; Thermo Fisher Scientific, Inc.), CD34-FITC (cat. no. CD34-581-01; Invitrogen; Thermo Fisher Scientific, Inc.), CD73-FITC (cat. no. 11-0739-42r; eBioscience; Thermo Fisher Scientific, Inc.), CD90-FITC (cat. no. 11-0903-82; eBioscience; Thermo Fisher Scientific, Inc.) or CD105-FITC (cat. no. MA1-19594; Invitrogen; Thermo Fisher Scientific, Inc.) was added. The cells were incubated in the dark for 45 min at 4˚C, following which, 400 µl of flow cytometry dyeing buffer (cat. no. PAB180076; Wuhan Beinlai Biotechnology Co., Ltd.) was added to each tube. The cells were subjected to flow cytometry (CytoFLEX S; Beckman Coulter, Inc.) and the results were analyzed using the CYEXPERT software (CXP Analysis 2.0; Beckman Coulter, Inc.).
Cells were cultured for 24 h at 37˚C, harvested, treated with 1 ml of pre-cooled PBS and centrifuged at 1,000 x g for 5 min at 4˚C. Subsequently, 10 µl of Annexin V-FITC and 10 µl of PI were added. The cell samples were then analyzed using flow cytometry as aforementioned. A one-step fluorescence compensation strategy was used to eliminate interference with the FITC channel (21 (link)).
Cells were cultured for 24 h at 37˚C, harvested, treated with 1 ml of pre-cooled PBS and centrifuged at 1,000 x g for 5 min at 4˚C. Subsequently, 10 µl of Annexin V-FITC and 10 µl of PI were added. The cell samples were then analyzed using flow cytometry as aforementioned. A one-step fluorescence compensation strategy was used to eliminate interference with the FITC channel (21 (link)).
7
Apoptosis Discrimination in Cell Cultures
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Discrimination between early and late apoptosis was performed as reported previously with certain modifications (32 (link)). Cells were incubated for 24, 48 or 72 h with or without different concentrations of liriopesides B (1×IC50 and 10×IC50). Adherent and floating cells were harvested and washed twice with PBS, and then suspended in 200 µl of 1X Binding Buffer, to which 10 µl Annexin V-FITC and 10 µl propidium iodide were added. After the cells were gently vortexed and incubated for 30 min in the dark, 300 µl of 1X Binding Buffer was added. The fluorescence of the cells was detected with a flow cytometer (FC500 MCL; Beckman Coulter, Inc.) and CXP Analysis 2.0 (Beckman Coulter, Inc.) was used to analysis the flow cytometry results.
8
Cell Cycle Analysis by Flow Cytometry
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Cells were cultured six-well plate at 5 × 105 cells/well. The cell supernatant was collected when the coverage rate in the experimental group increased to 80 %, ensuring that cells were in the logarithmic phase. Cells were washed twice with PBS and subjected to trypsinization. Cells were collected in a 5 ml centrifuge tube. Three compound perforations were prepared in each group and timed cycle tests were performed ensuring an adequate number of cells for computerized analysis, with at least 1,000,000 each time. Cells were collected after centrifugation at 2,000 rpm for 5 min and fixed with 70 % ethanol, which was pre-cooled to 4 °C overnight. The stationary liquid was abandoned by centrifugation at 2000 rpm for 5 min. Cells were washed with PBS twice. Cells were incubated in PBS containing 400 μl RNase (50 μg/ml) at 37 °C for 30 min. Cells were then stained with 800 μl PI (20 μg/ml) for 30 min in the dark at 37 °C. Cell cycle progression was analyzed using a flow cytometer (Beckman Coulter, Miami, FL, USA). During cell cycle analysis, gating and voltage were carefully set to exclude clumped cells and cell debris. The data were analyzed using CXP Analysis 2.0 (Beckman Coulter).
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