DeadEnd Fluorometric TUNEL Assay

  1. Turn on incubator (37C)
  1. Thaw Equilibration Buffer and DNase I buffer (optional) and then place on ice
  1. Aspirate media for chamber slide and wash 1x with 200ul PBS
  1. Add 100ul 4% paraformaldehyde in PBS to each well of a 8-well chamber slide. Incubate at 4C for 25min.
  1. Aspirate 4% paraformaldehyde and wash 1x with 200ul PBS
  1. Remove chamber from slide
  1. Wash slide by immersing in PBS for 5min at RT. Repeat. (All immersions done in Coplin jars)
  2. After this step, slides may be stored in PBS at 4C for up to 2 weeks.
  1. Permeabilize cells by immersing slide in PBS with 0.2% Triton X-100 for 10min at RT
  1. Wash slide by immersing in PBS for 5min at RT. Repeat
  1. For DNase treated positive control only (keep all other slides in PBS). Note: not needed if chemically induced positive control is used.
  2. Remove excess buffer by holding Kimwipe to edge of slide
  3. Add 100ul DNase I buffer to each well of a 2-well slide and incubate at RT for 5 min (make sure all cells are covered).
  4. Remove buffer by holding Kimwipe to edge of slide.
  5. Mix 100ul DNase I buffer with 10ul RQ1 DNase and add to 1 well of a 2-well slide. Incubate at RT for 10 min.
  6. Remove buffer by holding Kimwipe to edge of slide.
  7. Wash slide by immersing in ddH2O for 1min at RT. Repeat 3 times.
  8. From this point on, use a separate jar for the positive control
  1. Blot excess buffer by holding Kimwipe to edge.
  1. Add 100ul Equilibration Buffer per half of a slide slide and incubate at RT for 5-10min (make sure all cells are covered)
  1. During incubation, for each half of a slide, prepare a reaction mixture consisting of 45ul Equilibration buffer, 5ul nucleotide mix and 1ul rTdT Enzyme. For negative control, substitute ddH2O for rTdT. Store mixture on ice and protected from light.
  1. Master Mix ( x)= ul Eq. Buffer + ul nucleotides + ul rTDT
  1. Remove excess buffer by blotting edge with Kimwipe.
  1. Apply 50ul reaction mixture to each half of a slide. Cover with a cover slip (No bubbles!).
  2. For all subsequent steps, minimize exposure of slides to light
  1. Incubate in humidified 37C incubator for 60 min (Cover box with foil)
  1. Remove SlowFade Gold with DAPI from freezer and equilibrate to RT
  1. Remove coverslips and stop reaction by immersing slide in 2x SSC for 15min at RT (Cover jars with foil)
  2. For 80ml 2x SSC (40ml/jar), mix 8ml 20x SSC with 72ml ddH20
  1. Wash slide by immersing in PBS for 5min at RT. Repeat twice. (Cover jars with foil)
  1. Remove excess buffer by blotting edges with Kimwipe.
  1. Place 1 drop of anti-fade reagent in the center of each half and place coverslip on top (No bubbles!) If necessary, secure coverslip with clear nail polish. Let slides sit for at least 20min before visualizing (keep protected from light)
  1. View GFP using the green fluorescence filter, DAPI using the blue fluorescence filter and CF568 using red fluorescence filter.
  1. Stores slide covered in foil at 4C

Current as of 12-15-14

10x Phosphate Buffered Saline, pH 7.4 (for 1L):

80g NaCl

2g KCl

2g KH2PO4

11.5g Na2HPO4

Add ddH2O to 1L

Sterilize by autoclaving

4% Paraformaldehyde (for 100ml):

All steps done in fume hood

-Weight 4g paraformaldehyde

-Add 1x PBS to 100ml

-Heat to 65C for 2hr while stirring

-Let cool overnight while continuing stirring

-Store 4C

0.2% Triton X-100 in PBS (for 500ml):

-Add 1ml Triton X-100 to 500ml PBS

-If necessary, warm in water bath to get Triton into solution

DNase I Buffer (for 10ml):

400ul Tris HCl, pH 7.9

33ul 3M NaCl

60ul 1M MgCl2

50ul 2M CaCl2

9457ul ddH20

Filter sterilize. Store at -80C

Current as of 12-15-14

Current as of 12-15-14