Flow Cytometry Protocol.
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in this video we will illustrate the key steps in a flow cytometry.
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Experiment sample preparation cell fixation and permeable
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ization,
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immuno staining and analysis all of
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the solutions and re-agents you will need to prepare yourselves are shown on screen.
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Detailed information on how to prepare these solutions is available on our website.
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The first step will be to harvest your cells by gently scraping using
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PBS buffer containing five million Moller E D.
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T.
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A.
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Collect the cells and transfer to a 15-millimeter conical tube,
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then centrifuge and aspirate the super natan,
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wash the cells once with cold PBS and discard the Superintendent.
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Please skip the next two steps,
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fixation and permeability ation.
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If you're testing live cells re suspend the cells in fixation buffer and
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incubated room temperature for 30 minutes or overnight at four degrees Celsius
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centrifuge and remove the super natan,
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wash the cells twice with cold PBS and discard the super Needn't.
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Please skip the next step,
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permeability ation.
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If your protein of interest is extra cellular.
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Re suspend the cells in the permeability.
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Ation buffer and incubated at room temperature for 10 to 20 minutes After
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removing the super natan re suspend the cells in PBS and alec watt
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1 to 2 million cells into each test tube containing the blocking buffer,
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incubate on ice for 30 minutes and discard super nations add
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primary antibody at the appropriate dilution to the test tubes and incubate for 30 minutes on
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ice,
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centrifuge and discard the super Needn't wash the cells three times by
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centrifuge Gatien using fax buffer and remove the.
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Super natan after the last wash.
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If your primary antibody is directly linked to a floor a chrome,
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you can skip this step.
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Otherwise re suspend the cells in your fluorescent dye.
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Conjugated secondary antibody diluted to the appropriate concentration,
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incubate on ice for 30 minutes in the dark,
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wash the cells three times with fax buffer.
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Finally re suspend the cells in cold one X.
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PBS and analyze on the flow saitama ter as soon as possible.
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For more details and troubleshooting,
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please visit our website at origin dot com slash support slash
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protocols.