Fortessa x 20 flow cytometer

For measurement of relative intracellular free calcium concentration ([Ca²⁺]_i), RBC-depleted splenocytes (1E7/mL in complete medium containing 2% FCS) were stained with anti-B220 and Fab anti-mouse IgG (H+L), while being loaded with 5 μM indo-1 acetoxymethyl ester (INDO-1 AM, Thermofisher) according to the manufacturer’s protocols. Both Indo loading and flow cytometry were performed at room temperature (RT; ~22°C). After being washed once in medium, cells were resuspended at 5E6 cells/mL in RT medium in 500 μL aliquots. Indo-1 was excited with a 355 nm UV laser, and Ca²⁺-bound indo-1 was detected with a 379/28 bandpass filter; unbound indo-1 was detected with a 524/40 bandpass filter. Induced changes in relative intracellular free calcium concentration ([Ca²⁺]_i) was determined by calculating the ratio of Ca²⁺ bound/unbound indo-1 signals over time. After data were acquired for 30 s to establish [Ca²⁺]_i baseline, cells were stimulated with the indicated dose of goat F(ab’)₂ anti-mouse IgM, rabbit F(ab’)₂ anti mouse IgG (H+L), goat F(ab’)₂ anti-human IgM Cμ5, or rat anti-mouse IgM [B-7–6] (JacksonImmuno) and data were collected for an additional 2 min 30 s. The relative [Ca²⁺]_i was measured using a Fortessa X-20 flow cytometer and analyzed with FlowJo software.