Titration of Tetramers

Experiment to Show the Effect of Titrating MHC class I Tetramer on Non-specific Staining

[Method] [Gating] [Tetramer Dilution Effect] [Elimination of Non-specificity]

Method

The following protocol was used to show the effect of titrating the MHC class I tetramer HLA-A*0201/GILGFVFTL on the amount of non-specific tetramer binding.

Antibodies Used:

Channel	Specificity	Clone	Fluorochrome	Vendor	Volume
FL1	CD3	B9.11	FITC	Immunotech	20ul
FL1	CD3	UGHT1	FITC	Immunotech	20ul
FL2	CD3	UGHT1	PE	Immunotech	20ul
FL4	CD3	UGHT1	APC	Immunotech	10ul

Dilution of Tetramer:

MHC class I tetramer (HLA-A*0201/GILGFVFTL -PE) was diluted in FACS buffer (PBS plus 1% BSA). For the 1:100 dilution (one part tetramer to 100 parts sample, in this case whole blood), 4ul of concentrated tetramer was diluted with 6ul of FACS buffer. Therefore, 4ul of concentrated tetramer was added to 400ul whole blood (see below). Dilutions were made from the diluted tetramer for the 1:300, 1:900, 1:2700 and 1:8100 dilutions

Staining Cells:

Add 10ul of each tetramer dilution to an appropriate flow cytometry tube (negative control -- 10ul FACS buffer)
Add appropriate amount of each monoclonal antibody to appropriate tubes
Set up compensation tubes of only the CD3 antibody with the various fluorochromes
Add 400ul of fresh whole blood in EDTA and lightly vortex
Incubate tubes for 15 minutes at room temperature
Add 2ml of FACS lysing solution, vortex lightly and incubate 10 minutes at room temperature
Add 2ml of FACS buffer and spin down cells
Decant supernatant and resuspend cells in 2 ml FACS buffer and spin down cells
Decant supernatant, vortex lightly and fix with 200ul 1% paraformaldehyde

Gating

The following two graphs show the gates used in this experiment. The first gate was on the forward versus side plot and captured the lymphocyte population. The second gate was on the foward versus CD3 plot and captured the CD3 positive lymphocyte population.

Tetramer Dilution Effect

The following plots show the effect of titrating the tetramer on the amount of non-specific binding seen in the CD8 negative-Tetramer positive quadrent.

Elimination of Non-specificity

The following graph show the elimination of non-specific staining by the tetramer. The specific staining (tet+, CD8+) shows a gradual reduction of staining with titration of the tetramer, whereas, the non-specific staining (tet+, CD8-) is quickly eliminated.

Last updated September 3, 2005