WHOLE BLOOD STAINING FROM TAIL BLOOD.

•   Method #1: lyse first
•  Method #2: stain first

Grab the mouse (image) gently but firmly with your leading hand. With a new sharp sterile blade, nick the tail skin of the last 1/2 cm without touching the tail bone.
Dip the tip of the tail in the tube and gently milk the tail (image).
Allow any drop of blood to fall in the PBS. Maximum three large drops. Usually very little blood is sufficient.
Seal the tube with Parafilm and invert to mix the content.
Store the tube a RT for up to 2 hours max.
Centrifuge at 2,000 rpm for 5 min and aspirate the supernatant.
Tail and toe cutting is allowed only within the first 10 days of life, at which time the amount of blood is insufficient to do a phenotype. Use this system reproducibly for mice 5 weeks or older.

Method #1: lyse first.

Resuspend in the few µl left by vortexing and add 200µl of the Erythrocyte Lysing Solution (Becton Dickinson, FACS Lysing Solution Cat 349202, 1x in water from a 10x concentrate). Mix well. Let stand for 10-15 minutes until lysed.

Add approx. 3 ml of serum-containing medium and centrifuge at 1500 rpm for 5 min.

Aspirate the supernatant and wash again with 1 or 2 ml medium+serum.

After having carefully aspirated most of the washing solution, vortex briefly to loosen up the cells and add the directly conjugated antibodies in 50µl volume. Mix well. Incubate for 20 min (membrane or cytoplasmic antigens).

Add approx. 3 ml of serum-containing medium and centrifuge at 1500 rpm for 5 min.

Aspirate the supernatant and wash again with 1 or 2 ml medium+serum. Add FACS analysis solution (w or w/o formalin) in the appropriate volume (min 200 ? max 500 µl).

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Method #2: stain first.

Vortex and resuspend in 50 µl of medium, containing 10mM EDTA and the antibodies. Incubate for 15-30 min.
 (for TCR Vß staining mix CD90/Thy-1-Pe 1:50 and TCR Vß-FITC 1:50)

Wash twice (top up with medium, spin and aspirate the supernatant twice).
After having carefully aspirated most of the washing solution, vortex briefly to loosen up the cells and add 100 µl of the Erythrocyte Lysing Solution (Becton Dickinson, FACS Lysing Solution Cat 349202, 1x in water from a 10x concentrate). Mix well.

Let stand for 15 min max at RT.
Wash once with medium, resuspend with 300 µl medium (+ 10% formalin if not to be analyzed the same day).

Make sure you stain a sample with single fluorochromes for compensation.

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