APOPTOSIS

  DNA Labeling by Propidium Iodide

MATERIEL :

-Propidium Iodide , ex. Sigma Ref. P4170. Prepare a 1mg/ml solution. Store at 4°C.

-RNAse : ex RNAse A Boehringer Ref. 109169, heat 15 mn at 100°C, Store at  -20°C

-Labeling solution : IP at 40 µg/ml added with RNAse at 100µg/ml in PBS, Store at 4°C.

 2.105 à 106 cells in 5 ml tube for FACS washed once

Dilute cell pellet in 750 µl of a mix with 75% ethanol / 25% distilled waterat room temperature for 1 to 2 mn

Centrifugate and dilute cell pellet in 500 µl solution with 40 µg / ml in PBS with 100 µg /ml of RNAse DNAse free.

incubate 1 to 2 h at 37° C

Let at 4°C and analyze tubes on cytometer few hours later (1 to 2 days at 4°C sometimes allow a better resolution of the apoptosis peak).

Cell cycle analysis is also available

NB: Propidium iodide is very toxic, wear gloves (and a mask to weigh), do not empty the tubes in the sink.

REFERENCE :

-J Exp Med 1994, 179:1789

-J Exp Med 1992, 176:1043