How to set up your staining group?
1. For single staining, you need
a. None(unstained control)
- for setting PMT and find autofluorescence
b. Fc blocking(all tubes, especially to immune cell, if need)
- to block the non-specific detection due to the Fc region of Ab binding to FcR in some cells.
c. Isotype-matched control
- to eliminate non-specific binding (Fc binding and fluorophore binding)
- for setting negative threshold
d. The group stained with the Ab you selected.
2. For double staining without interference, you need
a. None
b. Fc blocking (all tubes, if need)
c. Isotype-matched control (2 colors)
d. The groups stained with Abs you selected(2 colors)
For example, detect the T and B lymphocytes from the mouse splenic suspension:
3. For double staining with interference, you need
a. None
b. Fc Blocking(all, if need)
c. Iso(2 color)
d. True positive for two different fluorochromes to correct the overlapped fluorescence profiles.
- 2 single stained positive tubes, separated
- the dose of Ab applied to the compensation tube must be same of the test tube
- Enough positive and negative signals in one tube
- the same autofluorescence between compensation tube and test tube
- collect enough events(阳性细胞大约检测10k～20k）
e. The groups stained with the Abs you selected.
Under the replaced conditions with previous example:
Homework3:
