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8
Instruction Manual
7.4
Remove the wash solution and add to each well 100 µL permeabilization
solution. Mix well but gently, incubate for 10-20 minutes at room temperature
and proceed to step 8. for the click reaction.
8. EdU Detection
8.1
Prepare the click assay cocktail in the same order as described in table 5. If the
ingredients are not added in the order listed, the reaction will not proceed
optimally or might even fail.
Important: Once the assay cocktail is prepared, use it immediately, at least within
the next 15 minutes!
Material
Reaction buffer
Catalyst solution
Dye Azide (10 mM)
Buffer additive
(prepared in 4.1.2)
Total Volume
Table 5: Click assay cocktails.
8.2
Remove permeabilization solution from step 7.4 and add 100 µL of the click
assay cocktail to each well and mix well but gently to distribute the assay
solution evenly.
8.3
Incubate the click assay mixture for 30 minutes at room temperature. Protect
from light!
8.4
From the 10x rinse solution prepare a 1x rinse solution by applying following
table (table 6). Add the appropriate amount of PBS (1X) (see table 6) to the
component F and mix well (To prevent crystallization, keep component F at
room temperature at all times. If component F has crystalized, please warm up to
dissolve again. Please see also "cautions"). This additional wash step with this
special rinse buffer reduces unspecific, cell number dependent background
signal. After use, store any remaining solution at RT. When stored as directed,
this stock solution is stable for up to 6 months.
EdU HTS Kit
1 x 96 well plate
2 x 96 well plate
Table 6: Amounts of aqueous solution needed to dissolve the rinse buffer to the final work solution
Remove Click assay cocktail and wash the cells in each well twice with 150 µL
with the 1X rinse solution prepared above.
Component
C-
orange
D -
green
B -
red
E -
blue
-
Volume of 10X rinse buffer
2.9 mL
5.8 mL
Number of 96-well plates
1
9.635 mL
220 µL
55 µL
1.1 mL
11.01 mL
Dilution volume of 1X PBS
2
19.27 mL
440 µL
110 µL
2.2 mL
22.02 mL
26.1 mL
52.2 mL
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