After acquiring single cell suspension and Fc block, I usually stain using biotinylated-antibody together with other conjugated primary antibodies in one master mix for 30min at 4C, followed by wash with FACS buffer once, and then stain for conjugated streptavidin alone for another 30min, followed by wash twice with FACS buffer. The cells are ready to be analyzed.

The indirect staining protocol you mentioned using biotin-tagged antibody and PE-streptavidin for FACS (Fluorescence-Activated Cell Sorting) is a common approach to increase signal amplification and minimize background noise. Below is a step-by-step guide for the protocol, including the recommended incubation duration and temperature for optimal results:

Materials Needed:

Biotin-tagged primary antibody

PE-streptavidin (conjugated to phycoerythrin fluorochrome)

Cells of interest

Buffer (e.g., PBS, 1% BSA, 0.1% sodium azide)

Optional: Fc receptor blocking agent (e.g., FcR blocking reagent) to reduce non-specific binding of antibodies.

Protocol:

Prepare your cell sample by isolating and suspending the cells of interest in a suitable buffer. The exact buffer choice will depend on your experimental setup and the nature of your cells.

Optional: If using a Fc receptor blocking agent, add it to the cell suspension and incubate for 10-15 minutes at room temperature to block non-specific antibody binding to Fc receptors.

Add the biotin-tagged primary antibody to the cell suspension at the appropriate dilution. The optimal antibody concentration will vary based on the antibody affinity and the expression level of the target antigen. You may need to perform a titration experiment to determine the best concentration for your specific application. Incubate the cells with the primary antibody for 30 minutes to 1 hour at 4°C (on ice). This step allows the primary antibody to bind specifically to the target antigen on the cell surface.

After the incubation with the primary antibody, wash the cells 2-3 times with the buffer to remove any unbound or non-specifically bound antibodies.

Add the PE-streptavidin to the cells at the recommended dilution (as per the manufacturer's instructions) and incubate for 30 minutes to 1 hour at 4°C (on ice). The streptavidin will bind to the biotin-tagged primary antibody that is already attached to the cell surface.

After the incubation with PE-streptavidin, wash the cells 2-3 times with the buffer to remove any unbound PE-streptavidin.

Your cells are now ready for FACS analysis. Proceed with the standard FACS staining and data acquisition procedures.

Important Considerations:

Always include appropriate control samples in your experiment to account for background fluorescence and non-specific binding. Controls should include unstained cells, cells stained with isotype control antibodies, and single-stained cells with either the primary antibody or PE-streptavidin alone.

Keep the cells on ice or at 4°C during the staining and washing steps to minimize internalization and degradation of surface-bound antibodies.

If your cells are sensitive to cold temperatures, you can perform the incubations at room temperature, but keep in mind that this might increase non-specific binding and require optimization of the protocol.

https://www.bio-rad-antibodies.com/fluorescently-labeled-streptavidin.html