Tech Tip: Staining Membranes in Fixed Cells (CytoLiner^TM Protocol)

Procedure for adherent cells:

1. Remove culture medium from live cells and rinse cells twice with HBSS buffer
   with Ca²⁺/Mg²⁺
2. Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature.
   Note: Fixation concentration, time, and temperature may require optimization for different cell types or co-stains.
3. Rinse cells 3 times with PBS.
4. Permeabilize cells with 0.1% Triton® X-100 in PBS for 10 minutes at room temperature.
   Note: Permeabilization time, temperature, and detergent concentration may require optimization for different cell types or culture conditions. Using higher concentrations of Triton® X-100 or permeabilizing for more than 10 minutes at room temperature may result in dimmer and less specific staining.
5. Rinse cells 3 times with PBS.
6. Prepare 1X CytoLiner^TM Staining Buffer by diluting the 100X buffer with water at a ratio of 1:100.
   Note: Staining may be done in other buffers, such as PBS, but the staining may be less specific. For best results, use 1X CytoLiner^TM Staining Buffer.
7. Prepare CytoLiner^TM Dye staining solution by diluting the 500X CytoLiner^TM Dye in 1X CytoLiner^TM Staining Buffer at a ratio of 1:500 and immediately vortex to mix completely.
   Note: Prepare the staining solution within 10 minutes of use and mix well before adding to cells. Do not add concentrated CytoLiner^TM Dye directly to cells in buffer because this may result in uneven staining. Dye concentration may require optimization for different cell types.
8. Remove PBS from cells and add the 1X staining solution with enough volume to cover the cells completely.
9. Incubate for 10 minutes at room temperature, protected from light.
   Note: Staining time may require optimization for different cell types.
10. Remove the staining solution and rinse cells 3 times with PBS.
11. Proceed with imaging, or continue to blocking and staining with antibodies or other probes.
    Note: After the initial permeabilization step and CytoLiner^TM staining, all buffers used for blocking, antibody incubation, and washing must be detergent-free.
    Note: Staining with CytoLiner^TM after antibody staining will work, however, the background may be higher.

Procedure for cells in suspension:

1. Transfer up to 10⁶ cells to a microcentrifuge tube.
2. Pellet cells by centrifuging at 350 x g for 3 minutes.
3. Remove supernatant and resuspend pellet in 500 uL of PBS to wash.
4. Pellet cells and repeat step 3 for two washes total.
5. Pellet cells and resuspend in 100 uL of 4% paraformaldehyde in PBS. Incubate for 15 minutes at room temperature.
   Note: Fixation concentration, time, and temperature may require optimization for different cell types or co-stains.
6. Pellet cells and remove supernatant. Wash twice with 500 uL of PBS as in step 3.
7. Pellet cells and resuspend in 100 uL 0.1% Triton® X-100 in PBS. Incubate for 10 minutes at room temperature.
   Note: Permeabilization time, temperature, and detergent concentration may require optimization for different cell types or culture conditions. Using higher concentrations of Triton® X-100 or permeabilizing for more than 10 minutes at room temperature may result in dimmer and less specific staining.
8. Pellet cells and wash twice in 500 uL PBS as in step 3.
9. Prepare 1X CytoLiner^TM Staining Buffer by diluting the 100X buffer with water at a ratio of 1:100.
   Note: Staining may be done in other buffers, such as PBS, but the staining may be less specific. For best results, use 1X CytoLiner^TM Staining Buffer.
10. Prepare CytoLiner^TM Dye staining solution by diluting the 500X CytoLiner^TM Dye in 1X CytoLiner^TM Fixed Cell Staining Buffer at a ratio of 1:500 and immediately vortex to mix completely.
    Note: Prepare the staining solution within 10 minutes of use and mix well before adding to cells. Dye concentration may require optimization for different cell types.
11. Pellet cells and remove supernatant. Add 100 uL of CytoLiner^TM Dye staining solution and resuspend cells by gently pipetting up and down.
12. Incubate for 10 minutes at room temperature, protected from light.
    Note: Staining time may require optimization for different cell types.
13. Pellet cells and wash twice with 500 uL PBS as in step 3.
14. Resuspend cells in 100 uL PBS.
15. Proceed with imaging, or continue to blocking and staining with antibodies or other probes.
    Note: After the initial permeabilization step and CytoLiner^TM staining, all buffers used for blocking, antibody incubation, and washing must be detergent-free

    Note: Staining with CytoLiner^TM after antibody staining will work, however, the background may be higher.

Figure 1. Comparison of fixed cell staining with CytoLiner^TM Fixed Cell Membrane Stains versus the classic CellBrite® Cytoplasmic Membrane Dyes. In comparison to the CellBrite® lipophilic carbocyanine dyes, CytoLiner^TM Dyes show more uniform and reliable staining of paraformaldehyde-fixed, mildly-permeabilized cell membranes. Paraformaldehyde-fixed cells were permeabilized for 10 minutes at room temperature with 0.1% Triton® X-100 in PBS, then rinsed with PBS and stained with CellBrite® Dyes in PBS or CytoLiner^TM Stains in 1X CytoLiner^TM Buffer for 10 minutes at room temperature, then rinsed with PBS. Imaged by confocal microscopy; scale bar: 20 um.

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