Eight thousand U2OS cells per well were seeded in a 96-well plate. After 8 hours of treatment, supernatant and cells were collected and transferred into a V-shape 96-well plate and centrifuged at 12,000 rpm for 5 min. Then, cells were incubated with diamidino-2-phenylindole (DAPI 0.5 µg/mL, #62248, Thermo Fisher Scientific) and with 3,3’-dihexyloxacarbocyanine iodide (DIOC 20 nM, #D273, Thermo Fisher Scientific) for 20 min at 37°C. Samples were analyzed using a CyAn ADP cytofluorometer (Beckman Coulter, Brea, California, USA) coupled to a HyperCyt loader (Intellicyt, Albuquerque, New Mexico, USA).
Hypercyt loader
Manufactured by Intellicyt
Sourced in United States
The HyperCyt loader is a modular instrument designed to automate the sample handling and analysis process for high-throughput flow cytometry applications. It functions as an integrated sample loading system, capable of accommodating a variety of sample containers and seamlessly interfacing with compatible flow cytometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Cyan adp cytofluorometer, by Beckman Coulter (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dioc 20 nm, by Thermo Fisher Scientific (1 mentions)
Biomek fxp, by Beckman Coulter (1 mentions)
Diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Live dead fixable dead cell stain, by Thermo Fisher Scientific (1 mentions)
Macsquant cytometer, by Miltenyi Biotec (1 mentions)
4 protocols using hypercyt loader
1
Apoptosis Imaging in U2OS Cells
2
Fully Automated Multimodal Cytometry
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All experimental steps were conducted on the PACRI HTS cell biology platform that integrates a Biomek FXP automated liquid handling workstation (Beckman–Coulter, Fullerton, CA, USA), a multidrop combi automated dispenser (ThermoScientific, Whaltam, MA, USA), a Cytomat6000 automated cell culture incubator (ThermoScientific), 3 ImageXpress Micro XL automated microscopes (Molecular Devices, Sunnyvale, CA, USA), a CyAn ADP cytofluorometer (Beckman–Coulter), a Hypercyt loader (Intellicyt, Albuquerque, NM, USA) and a Motoman HP3JC industrial robot (Motoman, West Carrollton, OH, USA). A fully automated workflow entailing cell culture, drug treatment, sample preparation and cytofluorometric or microscopic acquisition was generated by means of the SAMI automation control software (Beckman–Coulter). Multiplex assays were prepared in parallel for microscopic and cytofluorometric acquisition.
3
Quantification of CALR Expression in U2OS Cells
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Eight thousand U2OS wild-type cells per well were seeded in a 96-well plate. After 6 hours of treatment, cells were collected and transferred into a V-shape 96-well plate and centrifuged at 12,000 rpm for 5 min. Supernatant was removed and cells were incubated for 30 min at 4°C with primary rabbit monoclonal antibody against CALR (1:100 diluted in 1% BSA). Cells were washed, centrifuged at 12,000 rpm for 5 min and supernatant was removed. Cells were incubated with secondary AlexaFluor®488 goat anti-rabbit IgGs (1:1,000 diluted in BSA 1%) for 30 min at 4°C. Cells were washed, centrifuged at 12,000 rpm for 5 min and supernatant was removed. Finally, diamidino-2-phenylindole (DAPI, #62248, Thermo Fisher Scientific) was added before the analysis (1:400). Samples were analyzed using a CyAn ADP cytofluorometer (Beckman Coulter, Brea, California, USA) coupled to a HyperCyt loader (Intellicyt, Albuquerque, New Mexico, USA).
4
Detecting Cell Surface Calreticulin
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U2OS wild‐type cells were seeded at 8,000 cells per well in 96‐well plates. The cells were treated for 6 h, and the drug was washed out. Twenty‐four hours later, the cells were collected and ecto‐CRT was detected by immunofluorescence staining. Cells were incubated for 30 min at 4 °C with 1:100 primary antibody specific for CALR and then washed and further incubated with 1:500 secondary Alexa Fluor‐488‐coupled anti‐rabbit antibody for 30 min at 4°C. Finally, 4′, 6‐diamidino‐2 phenylindole dihydrochloride (DAPI, # D1306, Thermo Fisher Scientific) was added before flow cytometric analysis. Samples were analyzed using a CyAn ADP cytofluorometer (Beckman Coulter, Brea, CA, USA) coupled to a HyperCyt loader (IntelliCyt, Albuquerque, NM, USA). Alternatively, after incubation with primary antibody, cells were stained with the LIVE/DEAD Fixable dead cell stain (Thermo Fisher Scientific) and fixed with 3.7% formaldehyde. After staining with the secondary antibody, they were acquired with a MacsQuant cytometer (Miltenyi Biotec). Using FlowJo v10 software (TreeStar, Inc.), the percentage of CALR+ cells among DAPI− cells is quantified.
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