( bProtocol 1. Wash ...)Detection of antibody-stained cell,,,surface and intracellular protein targets,,,with the Agilent 2100 bioanalyzer

b>Protocol
1. Wash cells once in dye loading buffer after harvesting.
2. Stain cells with calcein (up to 5 x 106/ml, 0.5 M calcein, 15 minutes, 37 C) in dye loading buffer.
3. Wash once in staining buffer.
4. Stain cells (1 x 106) in a total volume of 100 l (80 l staining buffer and 20 l antibody) for 25 minutes on ice in the dark. For more cells (up to 5 x 106), adjust amount of antibody but don't increase total volume of 100 l.
5. Wash twice with 1 ml staining buffer.
6. Optional step if cells are to be stored overnight and measured later: Resuspend cells well at a cell density of 1 x 106 cells /ml in PBS. Make sure there are no cell clumps in the suspension. Add 70 l paraformaldehyde (16 %-solution) while stirring. Incubate for 10 minutes at room temperature (RT) and store sample at 4 C in the dark.
7. Resuspend cells in cell buffer at 2 x 106 cells/ml and load onto the chip.
b) Intracellular GR staining of H4 hepatocytes
Reagents
BD FACS Permeabilizing Solution 2 (500 tests) Alternatively, use Triton-X100 and Paraformaldehyde 16 % for fixation and permeabilization.
Anti rat GR antibody
Phycoprobe AP Anti-Mouse IgG
SYTO16
Staining buffer (PBS, 2 % BSA, 0.05% NaN3)
Protocol
1. Harvest cells and adjust cell density to 1 x 106 cells /ml in PBS.
2. Add 1.5 l SYTO16/ml and incubate 10 minutes at 37 C in the dark.
3. Wash cells in 2 ml staining buffer and centrifuge (500 x g, 5 minutes).
4. Resuspend cells in 500 l 1x BD permeabilizing solution (dilute in distilled water) and incubate 10 minutes at RT.
5. Two alternative steps, if permeabilization and fixation
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