I am using a Tecan plate reader to develop a high throughput fluorescent Ca imaging assay for measuring Thapsigargin stimulated calcium influx in human fibroblasts. The cells are seeded in clear bottom 96-well black plate and loaded with 100ul of Fluo-4 NW dye in assay buffer (HBSS + HEPES + probenecid) and incubated at 30min/37oc then 30mins/RT.
I notice that addition of 50ul of distilled water alone increases fluorescence by 20% above baseline values and even 10ul water increases fluorescence by 6%. The addition of Thapsigargin increases fluorescence by further 30% but I am sure the assay is losing sensitivity because of this volume effect. Any help or advice would be greatly appreciated.
Have you ever tried water with physiological osmolarity and pH instead of Dh2o? I guess this may change something in terms of Ca++ flux. Maybe worth the try.
Your undergraduate education should have told you that adding water reduces osmolarity and causes cells to swell. Since fluo-4 is not a ratiometric method this would be a serious problem. Unless you are deliberately studying osmotic effects, never use non-isotonic solutions.
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