Protocols

 

Cell wall pulse label with amine reactive dyes

The original tube (1mg) should be stored in -20°.
Suspend 1mg Alexa 488 in 500µl sterile water, mixed, divided into 12.5µl aliquots, and lyophilized. Each aliquot contains 80µg Alexa 488. Store in -80C.
To use the dye, reconstitute an aliquot in 5-10µl anhydrous DMSO, mix well, and use immediately. If the dye does not dissolve entirely, let the tube sit for 1-2 minutes and mix again– the dye should go fully into solution.
Staining protocol:
  1. Spin down log phase culture at 2000 rpm for 4 minutes.
  2. Remove supernatant and resuspend in 10ml PBS + 0.2% Tween 80 (PBST).
  3. Spin down at 2000 rpm for 4 min.
  4. Remove supernatant and resuspend in 500µl PBST.
  5. Add dye aliquot, flick to mix, and immediately spin down. Do not let the dye incubate on cells.
  6. Remove supernatant and resuspend in growth media or PBST.
 

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