Rare-event sorting by fixed-time flow cytometry based on changes in intracellular free calcium

Area of Science:

• Cell biology
• Biotechnology
• Neuroscience

Background:

• Intracellular calcium ([Ca2+]i) increases are key indicators of cellular responses to neurotransmitters.
• Identifying and isolating rare cell populations with specific responses is challenging in heterogeneous cultures.

Purpose of the Study:

• To evaluate fixed-time flow cytometry as a sensitive method for sorting rare neurotransmitter-responsive cells.
• To enrich specific functional cell types from mixed cell populations.

Main Methods:

• NIH/3T3 mouse fibroblasts were stimulated with a neurotransmitter cocktail.
• Fixed-time flow cytometry was employed to sort cells exhibiting intracellular calcium increases.
• Aseptic sorting and subsequent cultivation were used to enrich responder cell populations.

Main Results:

• Enrichment of serotonin-responsive NIH/3T3 cells from an initial frequency below 3 x 10(-4) to over 60% was achieved.
• Sorting of NIH/3T3 cells transfected with rat pituitary DNA yielded clones responsive to oxytocin or substance P, with initial frequencies below 2 x 10(-5).

Conclusions:

• Fixed-time flow cytometry is a highly sensitive technique for isolating very rare functional cells.
• This method enables the enrichment of specific cell subpopulations based on neurotransmitter-induced calcium signaling.