BD PharmingenTM Indo-1 AM

Description

Intracellular calcium response of Jurkat cells to stimulation with A23187. Jurkat cells were stained with 5 muM BD Pharmingen Indo-1 AM Cat. No. 565879 in DPBS with calcium and magnesium for 30 minutes at 37C. Cells were pelleted once and further incubated in complete medium for 30 minutes to allow complete hydrolysis of AM moieties. Cells were then pelleted and resuspended in DPBS with calcium and magnesium and analyzed using an BD LSRFortessa Cell Analyzer System with a 355 nm laser, 379/28 calcium bound and 515/30 calcium free bandpass filters, and a 450 dichroic mirror between. After 30 seconds of acquisition, the sample tube was briefly removed from the cytometer to facilitate addition of 1 muM calcium ionophore A23187 Sigma Aldrich, Cat. No. C7522 . Ratio: Bound/Free was calculated as the mean fluorescence intensity from the calcium bound channel Left Plot divided by the mean fluorescence intensity of the calcium free channel Middle Plot , and FlowJo Kinetic analyzer FlowJo, LLC was used to plot the average ratio versus time Right Plot . Jurkat cells show an increase in intracellular calcium concentration with addition of calcium ionophore A23187. Intracellular calcium response of Jurkat cells to stimulation with A23187. Jurkat cells were stained with 5 muM BD Pharmingen Indo-1 AM Cat. No. 565879 in DPBS with calcium and magnesium for 30 minutes at 37C. Cells were pelleted once and further incubated in complete medium for 30 minutes to allow complete hydrolysis of AM moieties. Cells were then pelleted and resuspended in DPBS with calcium and magnesium and analyzed using an BD LSRFortessa Cell Analyzer System with a 355 nm laser, 379/28 calcium bound and 515/30 calcium free bandpass filters, and a 450 dichroic mirror between. After 30 seconds of acquisition, the sample tube was briefly removed from the cytometer to facilitate addition of 1 muM calcium ionophore A23187 Sigma Aldrich, Cat. No. C7522 . Ratio: Bound/Free was calculated as the mean fluorescence intensity from the calcium bound channel Left Plot divided by the mean fluorescence intensity of the calcium free channel Middle Plot , and FlowJo Kinetic analyzer FlowJo, LLC was used to plot the average ratio versus time Right Plot . Jurkat cells show an increase in intracellular calcium concentration with addition of calcium ionophore A23187. Show More Intracellular calcium response of Jurkat cells to stimulation with A23187. Jurkat cells were stained with 5 muM BD Pharmingen Indo-1 AM Cat. No. 565879 in DPBS with calcium and magnesium for 30 minutes at 37C. Cells were pelleted once and further incubated in complete medium for 30 minutes to allow complete hydrolysis of AM moieties. Cells were then pelleted and resuspended in DPBS with calcium and magnesium and analyzed using an BD LSRFortessa Cell Analyzer System with a 355 nm laser, 379/28 calcium bound and 515/30 calcium free bandpass filters, and a 450 dichroic mirror between. After 30 seconds of acquisition, the sample tube was briefly removed from the cytometer to facilitate addition of 1 muM calcium ionophore A23187 Sigma Aldrich, Cat. No. C7522 . Ratio: Bound/Free was calculated as the mean fluorescence intensity from the calcium bound channel Left Plot divided by the mean fluorescence intensity of the calcium free channel Middle Plot , and FlowJo Kinetic analyzer FlowJo, LLC was used to plot the average ratio versus time Right Plot . Jurkat cells show an increase in intracellular calcium concentration with addition of calcium ionophore A23187.