Protocol for studying extracellular and intracellular proteins

Protocol for studying extracellular and intracellular proteins

Materials and equipment:

• Cell dissociation agent (e.g., trypsin) – for adherent cell lines only

• Cell culture medium containing FBS or trypsin inhibitors

• PBS or HBSS

• Benchtop centrifuge

• Fixative (e.g., 3% (w/v) Paraformaldehyde (PFA))

• Permeabilization solution (e.g., 0.1% (w/v) saponin or 0.1% (w/v) Triton X-100)

• Primary and secondary antibodies

• Flow cytometer

Experimental procedure:

1. Adherent cells:

1. Remove medium and wash cell monolayer with PBS

2. Add dissociating agent (e.g., trypsin) and incubate at 37°C for 5 min or until cells detach from the cell culture dish.

3. Add serum-containing medium or trypsin inhibitors to inactivate dissociating agent.

4. Transfer cells into a microcentrifuge tube.

2. Suspension cells:

1. Transfer cells into a microcentrifuge tube.

2. Pellet cells by centrifugation (300 G’s, 5 min at room temperature).

3. Remove medium and resuspend in PBS or HBSS.

4. Count cells and take 1 million (10⁶) cells per condition:

5. Unstained sample – for establishing autofluorescence levels

6. Sample stained with antibody A

7. Sample stained with antibody B

8. Sample stained with both antibodies

Note : Consider an isotype control to compensate for non-specific binding with a species-matched antibody.

Note : Fluorophores conjugated to antibodies A and B have to have different excitation spectra in order to distinguish their staining. Please refer to section 4 for tips on multicolor design experiments.

Optional: fix cells with a fixative agent (e.g., incubate with 3% (w/v) paraformaldehyde (PFA) solution for 20 min at room temperature). Centrifuge as in point 3 and wash cells with PBS.

Note: This step is recommended for staining intracellular targets.

3. For intracellular targets, permeabilize cells.

• For cytosolic protein staining, use milder detergents – e.g., incubate with 0.1% (w/v) saponin solution for 5-10 min.

• For nuclear protein staining and proteins inside cellular organelles, use harsher detergents – e.g., incubate with 0.1% (w/v) Triton X-100 solution for 5 min. Centrifuge as in point 3 and wash cells with PBS or HBSS.

4. Add antibody A and B and incubate for 20 min at room temperature (include milder detergent if it was used for permeabilization).

5. Pellet cells by centrifugation (300 G’s, 5 min at room temperature). Wash cells twice with PBS or HBSS.

6. If using unlabeled primary antibodies, incubate with secondary antibodies for 20 min at room temperature. Repeat step 7.

Note: When using secondary antibodies, primary antibodies have to be raised in different species to distinguish their staining. This is not a prerequisite when using fluorophore-conjugated primary antibodies because no secondary antibodies are needed for detection.

7. Analyze cells on a flow cytometer.