Intracellular Free Calcium Lab Report

Intracellular free calcium levels: Intracellular free calcium levels were estimated according to the method of Luo and Shi (Luo and Shi, 2005). Briefly, hippocampi were isolated from mice brains and subjected to hippocampal cells isolation as described later. The cells obtained were incubated with Fura-2 AM at 37°C with gentle shaking. The fura-2 loaded suspension was centrifuged for 10 minutes, pellet was washed once with Ca2+-free buffer and was centrifuged again. Aliquots of the washed suspension were diluted with buffer and pre-incubated for 6 min at 37°C. The intracellular free calcium concentration was determined by measuring the fluorescence at 340nm/380nm excitation and 510 nm emission. Maximum fluorescence (Fmax) was measured by lysis with 20% SDS or 2% triton X-100, followed by the addition of 0.5M EGTA (pH 8.0) to obtain minimal fluorescence (Fmin).
Calcium levels were measured according to the formula (The Grynkiewicz equation):
[Ca2+]i (nM) = Kd × [(R – Rmin)/Rmax – R)] × Sfb
Where, Kd (for Ca2+ binding to fura-2 at 37°C) = 225 nM, R = 340/380 ratio, Rmax = 340/380 ratio under Ca2+-saturating conditions, Rmin = 340/380 ratio under Ca2+-free conditions, and Sfb = ratio of
The hippocampus was dissected and minced using sterile scalpel. The minced tissue was placed in Dulbecco’s minimal essential medium (DMEM) and cell suspension was made gently using pestle in a 1.5 ml microcentrifuge tube. To this suspension appropriate amount of collagenase was added and incubated at optimum temperature i.e. 37°C for 25 minutes, mixing intermittently. The cells were dispersed gently by trituration, DNAse was added to stop the lysis reaction (final concentration 40µg/ml) and the cell suspension was filtered using 100 µm cell strainer, centrifuged at 500 g for 10 minutes. The supernatant was discarded and the pellet was resuspended in DMEM and cells were counted using