Apoptosis Detection

From Genome@Yale

Agilent Protocol

Annexin V-Biotin Kit (EMD, part #PF036)
Calcein-AM (EMD, 80050-600)
Cy5-Streptavidin (GE, PA45001)

following are for one sample (or 6 samples)
Solution A
   5uL (30uL) Medium Binding Reagent
   0.25uL (1.5uL) Annexin V-biotin

Solution B
   1X binding buffer (diluted from 5X with dH2O)

Solution C
   250uL (1.5mL) Solution B
   0.25uL (1.5uL) 1ug/uL Cy5 Streptavidin
   0.25uL (1.5uL) 5mM Calcein-AM

Technical note: prevent cells from clumping
   do not tap or hit the flask during trypsin treatment
   do not over trypinize the cells
   proceed immediately to analyze the cells after harvest, staining and washing

1. wash cells with 2mL PBS
2. add 200uL 0.25X Trypsin/EDTA
3. add 1mL media + 10% FBS and transfer the cells to eppendorf tubes
4. spin cells at 400rcf for 2 minutes at room temp
5. resuspend cells in 0.2mL media + 10% FBS
6. add 4uL Solution A
7. incubate 10 minutes at room temperature with gentle mixing (rotator)
8. spin cells at 400rcf for 2 minutes at room temp
9. remove media
10. resuspend cells in 200uL Solution C
11. spin cells at 400rcf for 2 minutes at room temp
12. carefully remove all liquid
13. resuspend cells in 30uL cell buffer by gentle tapping and mixing
14. analyze the cells on bioanalyzer