- Neutralize the remaining trypsin by adding 4.5 mL of cell
culture media. - Resuspend the cells by pipetting gently. Make sure there are no
cell clumps (seeNote 13). - Transfer 1 mL of the cell suspension to a 1.5 mL microcentri-
fuge tube and pellet the cells by centrifugation at 200gfor
5 min at 4C. - Aspirate the media and gently resuspend the cell pellet in 1 mL
of 1PBS. Make sure there are no cell clumps. - Count the cells.
- Pellet the required number of cells necessary for micropora-
tion (generally 50,000 cells per microporation) by centrifuga-
tion at 400gfor 5 min at 4C(seeNote 14). - Aspirate the PBS and resuspend the cell pellet in resuspension
buffer R at 5000 cells perμL. - Add 2μL of sample containing MBs and IRdye®800-labeled
dextran for every 10μL of cells such that the final concentra-
tions of MBs and IRDye®800-labeled dextran are 5 and 1μM,
respectively. - Gently mix the cells with MBs by pipetting.
- Microporate 10μL of the cell suspension (roughly 50,000
cells) at 1005 V with a 35 ms pulse width and 2 pulses total
(seeNote 14). - Transfer microporated cells to a microcentrifuge tube prefilled
with 1.5 mL of cell culture media. - Pellet the cells at 400gfor 5 min at 4C.
- Aspirate the media. Be careful not to disturb the cell pellet. Add
1.5 mL of cell culture media to resuspend the pellet. - Repeatsteps 15and 16 another two times (seeNotes 15and
16 ). - After the last wash, resuspend the cells in ~250μL of cell
culture media. - Seed the cells into a well of the 8-well chambered cover glass
previously coated with fibronectin (seeNote 17). - Image the cells using both Cy5 and IRDye®800 filter sets at
various time points.
3.4 Total
Fluorescence
Quantification
- Using Fiji, open a pair of images corresponding to the Cy5 and
IRDye®800 fluorescence of one object (microemulsion bubble
or cell). - Stack the Cy5 and IRDye®800 images using the function
Image>Stacks>Convert Images to Stack.
Optimizing Molecular Beacons 249