In 96 flat-wells plate, incubate 4x10 6 target cells (40 wells of 105 per well) with desired concentration of effectors (105 target cells per well). After incubation, collect the cell sample in 1.5 ml eppendorf tube, spin down, resuspend with 0.5 ml PBS in 1.5 ml eppendorf tubes, and add 55ul of lysis buffer for 20 min on ice (4oC). Centrifuge the eppendorf tubes in cold at 12,000 g for 30 minutes. Transfer the samples to new 1.5 ml eppendorf tubes and then extract the supernatant with 1:1 mixture of phenol:chloroform (gentle agitation for 5 min followed by centrifugation) and precipitate in two equivalence of cold ethanol and one-tenth equivalence of sodium acetate. Spin down, decant, and resuspend the precipitates in 30ul of deionized water-RNase solution (0.4ml water + 5ul of RNase) and 5ul of loading buffer for 30 minutes at 37oC. Also insert 2ul of Hindi III marker (12ul of Stock IV) on the outer lanes. Run the 1.2% gel at 5V for 5min before increasing to 100V.
Protocol II: SDS LysisBuffer
Add SDS lysis buffer to the incubated cell samples (prepared as in Protocol I).
Stock I:Triton X-100 Lysis Buffer 40 ml of 0.5 M EDTA 5 ml of 1 M TrisCl buffer pH 8.0 5 ml of 100% Triton X-100 50 ml of H2O
Stock II: SDS Lysis Buffer
Stock III: 1.2% Agarose Gel
Prepare a stock of 2 liter of 1X TAE (i.e., 2 liter + 40ml of 5X TAE). Add 2.4g of agarose power(1.2% agarose) to 200ml of 1X TAE solution and microwave for 4 min at high power. Then cool the gel to 50oC and add 25ul of ethium bromide before pouring it into the gel plate. Insert comb and let the gel polymerized.
Stock IV: Hindi III Marker (50 Kb lamda DNA) 4ul of Hindi III Marker 16ul of Deionized Water 4ul of Loading Buffer
Protocol II: DNA Fragmentation Assay via Dipheylamine
In 24-wells plate, incubate 5 X 106 targets with desired number of effectors. After incubation, transfer the samples to 15ml tubes, centrifuge for 30 s at 1500g, and resuspend in 5ml of lysis buffer (Stock IV) for 15 min on ice. Centrifuge the samples for 20 min at 27,000g to separate high-molecular-weight chromatin from cleavage products. Resuspend the pellet in 5 ml of buffer (stock V). Treat the supernatants and pellets with the diphenylamine reagent (Stock VI) and incubate at 370C for 16-24 hr before colorimetric assessment.
Stock IV: Lysis buffer at pH 8.0 5mM Tris-HCl 20mM EDTA 0.5% Triton X-100
Stock V: Buffer at pH 8.0 10mM Tris-HCl 1mM EDTA
Stock VI: Diphenylamine reagent (light sensitive) 1.5g of diphenylamine (steam-distilled) 100ml acetic acid (redistilled) 1.5ml of conc. sulfuric acid
On the day of usage, add 0.10ml of ag acetaldehyde (16mg/ml) to 20ml of the diphenylamine reagent.
Protocol III: DNA Fragmentation via 3H-TdR
5 X 106 target cells were labeled with 50µl of 3H-TdR (1 mCi/ml) overnight in 10 ml of media. The next day, the cells were washed 3X with 10ml of PBS and incubated in 10ml of media to chase out unincorporated cytoplasmic 3H-TdR. After incubating for 2 hrs, the cells were washed 3X with PBS and then used in lytic assay under the same conditions as the 51Cr release assay in 96 v-well plates. At the end of the assay, each well was treated with 20µl of 1.0% Triton-X on ice for 5 minutes, followed by centrifugation at 1500g in a Beckman T-J6 rotor for 15 minutes. 100µl of the supernatant were harvested from each well and counted in a scintillation counter. Total count was obtained by resuspending the cells prior to harvesting, and adding 0.1% SDS to solublilize the cells. The % 3H released was calculated with an equation analogous to that for %51Cr released.
Craig Walsh, Ph.D.