Computational protocol: Murine mesothelin: characterization, expression, and inhibition of tumor growth in a murine model of pancreatic cancer

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Protocol publication

[…] Panc02 cells were washed with DPBS and cells harvested by Trypsin-free Cell Dissociation Solution (Sigma-Aldrich). Cells were incubated in cold flow buffer (DPBS with 0.2 g % BSA + 0.01 % sodium azide) with 1:1000 of MBL B35 rat monoclonal antibody or normal rat IgG for background control. Following 30 minute incubation on ice, donkey anti-rat Cy5 conjugated secondary antibody was added at 1:100. Cells were incubated for an additional 30 minutes on ice, washes repeated and cells were fixed with 1 % electron microscopy grade paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) diluted in DPBS. Data were acquired on a BD LSR II (BD Biosciences) and data analysis was conducted using BD FACSDiva or FlowJo software. We found that trypsin cleaves mesothelin from the surface, so in experiments with adherent cells, cells were dissociated from the surface using trypsin-free solutions. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Neoplasms, Pancreatic Neoplasms