Hi, I'm now measuring the calcium flux assay with Fluo-8 AM dye in fungal cells (conidia and mycelia).

I've been using two calcium assay kits

1. Fluo-8 Calcium Flux Assay Kit - No Wash [ab112129], usually measured calcium with a microplate reader (96-black well plate) and followed protocol instructions.

2. Fluo-8 AM, green fluorescent calcium binding dye [ab142773], usually observing calcium with a fluorescence microscope.

1. Fluo-8 Calcium Flux Assay Kit - No Wash [ab112129], usually measured calcium with a microplate reader (96-black well plate) and followed protocol instructions.

For experimental conditions,

A1 - PBS treatment

A2 - 10 mM calcium treatment

A3 - 100 mM calcium treatment

A4 - 10 mM calcium treatment +4 uM TA

A5 - 100 mM calcium treatment + 4 uM TA

In a brief procedure,

1. 2.5 * 10^5 / 100 ul conidia was used.

2. Incubate 2h at 25°C.

3. Before measuring calcium levels with the microplate reader, 4 µM Thapsigargin was added to A4-5.

4. fluorescence data was collected every 5 seconds for 300 seconds. (Ex/Em = 485/528)

I only saw a decrease in fluorescence with the 100 mM calcium treatment, likely because high calcium levels are toxic to fungal cells. There was no difference between the PBS treatment and the 10 mM calcium treatment.

AND I don't know why the fluorescence doesn't increase in calcium treatment conditions...

2. Fluo-8 AM, green fluorescent calcium binding dye [ab142773], usually observing calcium with a fluorescence microscope.

For dye staining, I conducted three different experimental conditions.

1. Fluo staining 1h > wash 3 times > calcium treatment ( 5, 10, 50, 100, 200 mM) > wash 3 times > observed fluorescence microscope.

2. incubated calcium condition ( 5, 10, 50, 100, 200 mM) 1h > wash 3 times > Fluo staining 1h > wash 3 times > observed fluorescence microscope.

2. incubated calcium condition ( 5, 10, 50, 100, 200 mM) with Fluo dye 1h > wash 3 times > observed fluorescence microscope.

Like microplate reader results, I only observed a decrease in fluorescence with the 100, 200 mM calcium treatment. and there was no difference between the non-calcium treatment and the calcium treatment condition.

How can I make difference between the non-calcium treatment and the calcium treatment condition??