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.
- Spin down log phase culture at 2000 rpm for 4 minutes.
- Remove supernatant and resuspend in 10ml PBS + 0.2% Tween 80 (PBST).
- Spin down at 2000 rpm for 4 min.
- Remove supernatant and resuspend in 500µl PBST.
- Add dye aliquot, flick to mix, and immediately spin down. Do not let the dye incubate on cells.
- Remove supernatant and resuspend in growth media or PBST.