Immunofluorescence Staining Protocol (Cultured Cells)

1. Fixation:

• Wash cells with PBS.
• Fix in 4% paraformaldehyde for 10–15 min at room temp.
• Wash 3× with PBS.

2. Permeabilization:

• Incubate in 0.1–0.3% Triton X-100 for 5–10 min.
• Wash 3× with PBS.

3. Blocking:

• Incubate in 1–5% BSA or 10% normal serum for 30–60 min.

4. Primary Antibody:

• Incubate with primary antibody (in blocking buffer) for 1–2 hrs RT or overnight at 4°C.
• Wash 3× PBS.

5. Secondary Antibody:

• Incubate with fluorophore-conjugated secondary antibody (1 hr, RT, in dark).
• Wash 3× PBS.

6. Optional Nuclear Stain:

• Stain with DAPI (1 µg/mL, 5 min).
• Wash with PBS.

7. Mounting:

• Use anti-fade medium. Image with appropriate filters.

Cell Viability Using PI and Texas Red

Propidium Iodide (PI):

• Add PI (1–5 µg/mL) to live, unfixed cells.
• Incubate 5–10 min at RT, image immediately (red channel).
• PI stains non-viable cells (compromised membranes).

7-AAD if you are just interested in see cells with not broken membrane that can be associated with cell viability. Don't use impermeabilization or they obviously die. If you want to check early apoptosis use anexin-v. Look into apoptotic bodies with DAPI can give you an idea of advanced apoptosis. Or you can go down to stain activated Caspases both activators or effectors.

Chloe Sun

Propidium iodide (PI) cannot penetrate the intact cell membrane of live cells, so it can be used to distinguish live cells from dead cells; while Texas Red is only a fluorescent dye and does not have the ability to assess cell viability (i.e. it cannot detect cell viability status).

To facilitate the assessment of cell viability, you can also consider PI-based detection kits, such as: Live/Dead Cell Viability/Cytotoxicity Assay Kit (Calcein/PI) (MCE, HY-K1094)

https://www.medchemexpress.com/inhibitor-kit/viability-cytotoxicity-assay-kit-for-live-dead-cells-calcein-pi.html

The answer to this question comes from MedChemExpress (MCE) Technical Support.

[email protected]

Hi! There are many ways to assess cell viability. The simplest one is the trypan blue exclusion method — it even works with a standard light microscope. You can also use the classical combination of Hoechst (or DAPI) and Propidium Iodide. Hoechst is a vital dye that stains all nuclei in the culture, while Propidium stains only dead cells. In this case, of course, the culture must be alive and not fixed. If you add Annexin staining, you’ll also be able to distinguish apoptotic cells from necrotic ones.

There are also indirect metabolic assays — MTT, MTS, resazurin, CCK-8, or crystal violet — but these are more useful when you're expecting actual changes in the culture, rather than just checking how the cells are doing.