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Human Whole Blood Translocation experiment
O:\2009 Data\050209 G2A1 whole blood DM NFkB 6color
050209 G2A1 whole blood DM + LPS 6C 60X_17_48nofs_1.daf
050209 G2A1 whole blood DM 6C 60X_17_48nofs_1.daf
Background and purpose
NFkB translocation is an important indicator of blood cell activation, and is an important therapeutic target for the treatment of autoimmunity, cancer, and infectious diseases. To date, it has not been possible to robustly measure NFkB translocation directly in whole blood leukocytes. Here we show a simple method of quantifying NFkB activation in immunophenotypically-defined whole blood leukocytes using the ImageStream
1 – stimulate whole blood with 100 ng/mL LPS
2 – stain for surface markers on ice
PE-CD14, PE-TR-CD45, AF647-CD16, APCAF750-CD3
3 – Fix/lyse/perm and stain for NFkB (FITC) and DAPI
4 – ImageStream: collect 20,000 events at 60X
5 – Analyze leukocyte subsets for NFkB translocation
How many cells are needed – this slide illustrates the confidence in interpreting the extent of nuclear translocation in a sample exhibiting incomplete activation.
These are LPS stimulated human monocyte line THP-1 stained with FITC NFkB (green) and 7-AAD (nuclear dye, red). Histogram plots the per cell ‘degree of translocation’ score for the sample. As described in detail later, this is the ‘Similarity’ score for the NFkB / 7-AAD image pair.