#2   CRYSTAL VIOLET PROLIFERATION ASSAY B
      (alternative to MTT when using coculture inserts or larger
numbers of cells)


Protocol for a 24 well plate format


1.      Seed 2.5 - 5x 10^4 cells in 1ml media (if virus or other
treatments, treat cells on the following day) – 1 plate per timepoint


2.      Day 1, 3, 5, 7 or Day 1, 3, 6 timepoints:


a.      Aspirate media, wash cells with 1xPBS, aspirate (if there's virus
do this in the TC hood) – if you have inserts in wells, fix inserts in
a separate well.
b.      Add 500ul 10% buffered Formalin to cells
c.      Room temp 15 min (could store them dry at this point)- fume hood
d.      Add Crystal violet/water solution – Room Temp 15-30min
e.      Wash cells in sink with lots of water, slow flow not to detach cells
f.      Let dry inverted, then dry facing up
g.      Data: pictures or Measure absorbance


3.      Measuring absorbance at A595 or A600 (A630 is not good for dye
spectrum  anymore)

a.      After violet-stained cells are dried on plate, add 250ul 33%
glacial acetic acid using repeater pipette (in fume hood)
b.      Rock the plate until the acetic acid extracts the dye out of the
cells onto the solution
c.      Take a 96 well plate, and take 5ul from each 24well sample  place
in the 96well plate
d.      Then, add 150ul water to each 96well, mix
e.      Measure absorbance using plate reader, A550-600 ok, A630 not ok.
f.      Analysis of data is like for MTT or XTT curves (Day1, 3, 6 curve of growth)