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To use this antibody in FACS, are there any special steps that need to be taken?
Asked on May 23 2005
Thank you for your enquiry. The originator of ab9023 has passed along the following FACS protocol to use with this antibody. Example protocol: anti-cytokeratin M20 FACS analysis Materials: •Methanol -20 °C •PBS •PBS/BSA (1 mg/ml BSA in PBS) •Propidium/RNAse solution (20 µg/ml propidium iodide, 100 µg/ml RNAse in PBS) Antisera: Primary antibody •M20 (mouse anti-cytokeratin 8), 1:20 in PBS/BSA Secondary antibody •FITC conjugated anti-mouse Ig (DAKO F313 1:15 in PBS/BSA) Cell harvesting and fixation: •Harvest cells quantitatively by trypsinisation •Spin cell suspension for 5 min at 400 x g •Resuspend cell pellet in 200 µl PBS •Add under constant agitation 3.5 ml cold methanol (-20 °C) •Store cell suspension in Methanol in refrigerator until use Immunocytochemistry: •Spin methanol fixed cell suspension for 5 min at 400 x g •Discard supernatant and resuspend pellet in 1 ml culture medium •Spin cells at 400 x g •Incubate approximately 10^6 cells in 100 µl with the primary antibody for 1h at RT or overnight at 4 °C •Rinse twice with PBS/BSA •Incubate cells in 100 µl with FITC conjugated secondary antibody for 1 h at RT •Rinse with PBS/BSA •Resuspend cells in propidium iodide/RNAse solution Incubate for 15 min on ice prior to flow cytometry analysis
Answered on May 24 2005