EMT-6 hHER2+ cells were harvested with Versene and harvested by centrifugation (500 rcf) and resuspended in PBS with 10 mM MgCl2 and 1 mM ATP (Sigma Aldrich, A1852–1VL) 30 min at 37 °C in the presence of CK2α fusion proteins (1 μM) or vehicle. Cells were washed three times with cold 1% BSA in PBS (500 rcf, 5 min) and incubated on ice for 30 min with 100 nM scFv and StrepTactin-XT DY647 conjugate (IBA Lifesciences, 2-1568-050) at a 1:200 dilution in 1% BSA in PBS. Cells were washed two times with cold 1% BSA in PBS (500 rcf, 5 min), once with PBS (500 rcf, 5 min), and incubated for 15 min in PBS with Propidium Iodide Ready Flow (Thermo Scientific, R37169) live/dead cell stain. Cells were analyzed on a Beckman Coulter CytoFLEX flow cytometer.
Propidium iodide ready flow
Manufactured by Thermo Fisher Scientific
Propidium Iodide Ready Flow is a nucleic acid stain used to detect DNA content and cell cycle distribution. It is a ready-to-use solution that can be directly added to cell samples for flow cytometry analysis.
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Lab products found in correlation
3 protocols using propidium iodide ready flow
1
HER2+ Cell Analysis by Flow Cytometry
2
CK2α Binding and Live/Dead Analysis
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Cells were lifted from adherent monolayer culture with Versene (Fisher Scientific, 15-040-066) at 37 °C, harvested by centrifugation (500 rcf), and resuspended in PBS with 10 mM MgCl2 and 1 mM ATP (Sigma Aldrich, A1852–1VL) in the presence of cMyc-tagged CK2α fusion protein (1 μM). Cells were incubated at 37 °C for 30 min, washed three times with cold 1% BSA in PBS (500 rcf, 5 min), and incubated on ice for 30 min with anti-cMyc clone 9E10 AlexaFluor 647 conjugate (Thermo Fisher Scientific, MA1980A647) at a 1:100 dilution in 1% BSA in PBS. Cells were washed two times with cold 1% BSA in PBS (500 rcf, 5 min), once with PBS (500 rcf, 5 min), and incubated for 15 min in PBS with Propidium Iodide Ready Flow (Thermo Scientific, R37169) live/dead cell stain. Cells were analyzed on a Beckman Coulter CytoFLEX flow cytometer.
3
Antibody Binding Assay for HER2+ Cells
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EMT-6 hHER2+ cells were harvested with Versene and harvested by centrifugation (500 rcf) and resuspended in PBS with 10 mM MgCl2 and either 1 mM ATP (Sigma Aldrich, A1852–1VL) or 1 mM N6-phenylethyl-ATP (Biolog, P 012–05) for 30 min at 37°C in the presence of CK2α fusion proteins (1 μM) or vehicle. Cells were washed three times with cold 1% BSA in PBS (500 rcf, 5 min) and incubated on ice for 30 min with diluted serum (1:50) from immunized animals. Cells were washed three times with cold 1% BSA in PBS (500 rcf, 5 min) and incubated on ice for 30 min with goat anti-mouse IgG AlexaFluor 647 conjugate (Thermo Fisher Scientific, A28181) at a 1:200 dilution in 1% BSA in PBS. Cells were washed two times with cold 1% BSA in PBS (500 rcf, 5 min), once with PBS (500 rcf, 5 min), and incubated for 15 min in PBS with Propidium Iodide Ready Flow (Thermo Scientific, R37169) live/dead cell stain. Cells were analyzed on a Beckman Coulter CytoFLEX flow cytometer.
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