Titration of LD Dye Protocol

Titration of Live-Dead Stain

The goal of the L/D titration is slightly different. Unlike Ab titration we need to find a concentration that will provide us a good separation (Not maximum) between live and dead and same time keep the binding to the live cells lowest. These types of dyes called Amine reactive dye. This dye will bind to any protein it can find. This is why it is utmost import to use Protein free buffer for this assay. The dye will bind to the surface of the live cell but it will bind to dead cell many more folds as it has pores and more protein is accessible.

It is not necessary to use the same cell that will be used in real experiment. If possible one can use the same cell. Following protocol is based on cultured cell/PBMCs or Spleen cell.

  1. Harvest cell (10 million) in a FACS tube
  2. Spin at 400g for 5 minutes
  3. Remove supernatant
  4. Add 3 ml PBS, vortex briefly
  5. Spin at 400g for 5 minutes
  6. Remove supernatant
  7. Resuspend in remaining buffer
  8. Add 1 ml chilled (-20oC) 70% Alcohol in drop by drop manner while vortexing the cell
  9. Add another 9 ml chilled 70% Alcohol
  10. At this point can be stored for several years in -80oC (preferably in 10 aliquots)

At the day of the titration

Prepare cell

  1. Wash 4 million dead cells by adding 3 ml of cold PBS, vortex briefly
  2. Spin at 1000g for 5 minutes
  3. Remove supernatant
  4. Repeat the washing
  5. Collect in 800 µl of cold PBS
  6. Harvest the same live cells and wash with 400g as described as above
  7. Collect in 800 µl of cold PBS
  8. Mix the cells together, and distribute equally in 8 FACS tubes

Prepare Live-Dead dye

Reconstitute the powdered material in fresh DMSO as described by the manufacturer. Make sure you are using fresh unopened DMSO (preferably small glass ampules). Oxidized/used/opened DMSO changes the chemistry and decrease the potency of the dye many folds. Once reconstituted protect from light, keep at RT. Usually 1000-fold is the recommended concentration by the manufacturer.

  1. Add 0.4 µl of LD dye in 19.6 µl of PBS (make sure PBS is protein free). Add 10 µl of this LD buffer in one tube: 1000 fold dilution
  2. Add 10 µl of fresh PBS in the remaining 10 µl LD buffer, Add 10 µl of this LD buffer in one tube: 2000 fold dilution
  3. Repeat the process till you got 32,000 fold dilution.
  4. At this point you will have 6 stained tubes and keep one unstained tube
  5. Incubate for 15 minutes at RT in dark
  6. Add 3 ml of cold stain buffer (BD, 554657), quick vortex
  7. Spin at 1000g for 5 minutes
  8. Remove supernatant
  9. Repeat the washing
  10. Resuspend in 300 µl of stain buffer
  11. Determine what concentration you like to use.
  12. Based on this value prepare aliquots, make sure one aliquot will be good enough for one whole experiment.
  13. Store the aliquots in -20oC, in dark, do not freeze thaw aliquots, this will drastically decrease the potency.