5. Discard the labeling cell medium by decanting (or aspiration
from the cells growing on cover glass for immunofluorescence
microscopy), and quickly wash the plates three times in
quenching solution (not less than 1 mL per 3 cm
2 surface
area per wash). Pre-aliquot quenching solution into tubes and
rapidly, but carefully pour wash solutions to ensure quick and
efficient quenching of the reaction. Consider recruiting an
extra pair of hands and streamline this step, in order to minimize the time gap between proximity biotinylation and UV
cross-linking.
6. Leave quenching solution on cells growing on cover glasses,
which will not undergo UV cross-linking while handling the
other samples.
7. Decant quenching solution from cell plates for UV crosslinking and ensure that cells are covered by a liquid film to
avoid them from drying out. If necessary, add 750 μL (not
more, because cells will need to be scraped and collected into
1.5 mL tubes) of quenching solution to cells growing in plates,
remove lids, and cross-link cells with at least 0.15 J/cm
2 of
>310 nm UV light. (Note: Mark cell plates and not only the
lids, to avoid any switching of samples.)
8. While samples are cross-linking, return to aspirate the last
quenching solution wash, off the cells growing on cover glasses
for control immunofluorescence, and fix cells by fixative of
choice. After fixation, wash cells three times in PBS, and store
them in PBS at 4
C, for later further processing and imaging.
See Note 6 on fluorescence microscopy cell imaging.
9. When UV cross-linking is completed (on our instrument after
5 min) cover plates and place them on ice. Scrape the cells off
using a rubber policeman and collect them with the remaining
quenching solution into pre-chilled 1.5 mL tubes. Pellet cells
at 300 g, remove the supernatant, snap freeze cell pellets in
liquid N 2 , and store at À80
C.
3.3 Processing
of Cells from Control
Plates
1. Transfer tubes with the cell pellets from 6 cm control plates
from À80
C storage to ice and resuspend pellets in 300 μL of
cold RIPA buffer supplemented with 1 mM PMSF, 1Â protease inhibitors cocktail, 10 mM sodium azide, 10 mM sodium
ascorbate, and 5 mM Trolox (sodium ascorbate and Trolox
should be fresh and can be weighed and directly dissolved in
the appropriate amount of RIPA buffer).
2. Incubate the resuspended cells in RIPA buffer on ice for 2 min,
and then clear the extracts by centrifugation at 15,000 Â g for
10 min at 4
C. Carefully transfer the clarified extract without
disturbing the precipitated debris into new pre-chilled tubes.
290
Daniel Benhalevy and Markus Hafner
from the cells growing on cover glass for immunofluorescence
microscopy), and quickly wash the plates three times in
quenching solution (not less than 1 mL per 3 cm
2 surface
area per wash). Pre-aliquot quenching solution into tubes and
rapidly, but carefully pour wash solutions to ensure quick and
efficient quenching of the reaction. Consider recruiting an
extra pair of hands and streamline this step, in order to minimize the time gap between proximity biotinylation and UV
cross-linking.
6. Leave quenching solution on cells growing on cover glasses,
which will not undergo UV cross-linking while handling the
other samples.
7. Decant quenching solution from cell plates for UV crosslinking and ensure that cells are covered by a liquid film to
avoid them from drying out. If necessary, add 750 μL (not
more, because cells will need to be scraped and collected into
1.5 mL tubes) of quenching solution to cells growing in plates,
remove lids, and cross-link cells with at least 0.15 J/cm
2 of
>310 nm UV light. (Note: Mark cell plates and not only the
lids, to avoid any switching of samples.)
8. While samples are cross-linking, return to aspirate the last
quenching solution wash, off the cells growing on cover glasses
for control immunofluorescence, and fix cells by fixative of
choice. After fixation, wash cells three times in PBS, and store
them in PBS at 4
C, for later further processing and imaging.
See Note 6 on fluorescence microscopy cell imaging.
9. When UV cross-linking is completed (on our instrument after
5 min) cover plates and place them on ice. Scrape the cells off
using a rubber policeman and collect them with the remaining
quenching solution into pre-chilled 1.5 mL tubes. Pellet cells
at 300 g, remove the supernatant, snap freeze cell pellets in
liquid N 2 , and store at À80
C.
3.3 Processing
of Cells from Control
Plates
1. Transfer tubes with the cell pellets from 6 cm control plates
from À80
C storage to ice and resuspend pellets in 300 μL of
cold RIPA buffer supplemented with 1 mM PMSF, 1Â protease inhibitors cocktail, 10 mM sodium azide, 10 mM sodium
ascorbate, and 5 mM Trolox (sodium ascorbate and Trolox
should be fresh and can be weighed and directly dissolved in
the appropriate amount of RIPA buffer).
2. Incubate the resuspended cells in RIPA buffer on ice for 2 min,
and then clear the extracts by centrifugation at 15,000 Â g for
10 min at 4
C. Carefully transfer the clarified extract without
disturbing the precipitated debris into new pre-chilled tubes.
290
Daniel Benhalevy and Markus Hafner