Immunofluorescent Staining of Cell Surface Antigens for Flow Cytometric Analysis (FACS Analysis)
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Protocol A: Cell Preparation of Tissue Culture Cells
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Protocol B: Cell Preparation from Lymphoid Tissue
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Protocol C: Cell Preparation from Non-Lymphoid Tissue
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Protocol D: Isolation of PBMC from whole blood
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Protocol A: RBC Lysis of Mouse Splenocytes
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Protocol B: RBC Lysis of Mouse Blood
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Protocol C: RBC Lysis of Human Peripheral Blood
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Protocol D: Lysis of Human Blood for Flow Cytometric Analysis
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Protocol A: Cell Suspensions
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Protocol B: Human Lysed Whole Blood
Introduction
Precautionary Note
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Prior to using antibodies, do a quick spin to recover the maximum volume from the vials.
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We monitor our conjugated antibodies to make sure they are void of aggregation. Some protocols include a step of airfuging the antibody prior to staining to further clear aggregates.
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For optimal performance of fluorochrome conjugated antibodies, store at 4°C in the dark. Do not freeze.
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When staining, please note that fixation and/or delayed analysis may yield poor signal-to-noise for certain antigens. For best results, stain fresh mouse cells and analyze soon after staining.
Procedure Overview
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Making a single cell suspension from peripheral blood, lymphoid tissues or cultured cell lines
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Staining cells with antibodies (either as a directly fluorochrome-conjugated antibody or in successive steps of unlabelled antibody and fluorochrome-conjugated secondary reagents)
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Washing steps to remove all unbound reagents
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Running and analyzing on a flow cytometer
Note: In setting up a flow cytometry staining experiment it is important that all experimental conditions are optimized. Critical parameters include target cell, antibody titer and kinetics.
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