ICC Cell Smear Protocol for Suspension Cells

1. Adjust cells to a density of 1 X 10^6 cells/ml, then fix by adding 4% paraformaldehyde directly to culture media for 20 minutes at room temperature.

2. Pipette 1 ml of the fixed cell suspension into a 1.5 ml microcentrifuge tube and spin down cells for 30 seconds.

3. Remove supernatant and resuspend the cell pellet in 1 ml diH2O. Spin down at top speed for 30 seconds.

4. Remove supernatant and resuspend the cell pellet in 200 ul diH2O.

5. Add 5 ul of the cell suspension to a 22 mm x 22 mm poly-L lysine coated coverslip (~3 spots per slide), then smear the suspension using the side of a pipette tip.

6. Allow all of the liquid to evaporate by placing the coverslips on a hot plate (choose low heat setting!).

7. Check for salt crystals by placing the coverslip under a microscope.  Lightly wash the coverslip with di water if crystals are present.

8.  Place the coverslips in optical grade 6-well tissue culture plates for immediate use, or store at 2-8 degrees Celsius for up to 3 months for future experiments.

Fluorescent ICC staining protocol for cell smears

1.  Permeabilize cells as follows, depending on protein type:

a.  For detection of either nuclear or mitochondrial protein, add 1 ml TBS with 0.5% Triton X-100.  Incubate for 10 minutes at room temperature.
b.  If antibody is specific for detection of a cytoplasmic or membrane protein, add 1 ml PBS with 0.5% Tween-20 for 10 minutes at room temperature.

2. Take off permeabilization buffer and add 1 ml PBS plus 0.1% Tween-20, not letting the specimen dry out. Wash 3 times for 5 minutes before proceeding to blocking step.

3. To block nonspecific antibody binding, incubate in 10% normal serum from the host of the secondary antibody for 1 hour at room temperature. 

4. Add primary antibody (diluted in block: 10% normal serum or appropriate) and incubate overnight at 4 degrees Celsius.

5. Remove primary antibody and replace with PBS. Wash 3 x 5 minutes in PBS with 0.1% Tween-20.

6. Add secondary antibody (diluted in block: 10% normal serum) at appropriate dilution as per manufacturer’s recommendations.  Incubate for 1 hour at room temperature

7. Remove antibody and replace with PBS, wash 1 x 5 minutes in PBS with 0.1% Tween-20.  For second wash, add Hoechst 33258 to PBS at 1:25,000 and incubate for 10 minutes.  Wash a third time, adding phalloidin at 1:40 diluted in PBS with 0.1% Tween Tween-20 for 20 minutes (total of 3X5 minute PBS Tween-20 washes).

8. Carefully remove the coverslips from the wells and blot to remove any excess water. Dispense 1 drop of anti-fade mounting medium onto the microscope slide per coverslip. Mount the coverslip with the cells facing towards the microscope slide and tack edges down using clear nail polish.

9. Visualize using a fluorescence microscope and filter sets appropriate for the label used (please see list below). Slides can also be stored in a slide box at < -20 °C for later examination.

Fluorescent Dyes                λabs (nm)    λfl (nm)

 Atto390                                   390             479
Atto425                                   436             484
Atto465                                   453             508
Atto488                                   501             523
Atto532                                   532             553
Atto565                                   563             592
Atto590                                   600             627
Atto594                                   601             627
Atto633                                   629             657
Atto637                                   630             659
Atto655                                   663             684
Atto680                                   670             710
Atto700                                   700             725
DyLight 488                            493             518
FluoProbes®547H                  557             572
FluoProbes®647H                  653             675
FluoProbes®682                     690             709
FluoProbes®752                     748             772
AMCA                                      346              442
FITC                                        485              535
Rhodamine                              544              576
Texas Red                               595              615