You are studying the ability of your cultured cells to increase Ca == intracellu

Question

You are studying the ability of your cultured cells to increase Ca== intracellularly in response to a newly identified agent you have extracted from a soil fungus during a pharmaceutical screen. You are trying to identify HOW that increase in [ Ca==] is occurring, ie via entry of Ca from extracellular medium OR the ability of this novel agent to elevate intracellular [ Ca==] via activation of IP3 production. Outline THREE DIFFERENT approaches you would use, and the outcomes you would expect IF the increase in intracellular [ Ca==] was from extracellular sources or release of Ca== from intracellular stores.

Use the format below to describe your experimental strategy, and then the expected outcomes ( i.e., IF…..THEN…. )

Experimental Strategy

IF Ca++ from EXTRACELLULAR STORES

If Ca++ from INTRACELLULAR IP3-responsive stores

1)

2)

3)

Experimental Strategy

IF Ca++ from EXTRACELLULAR STORES

If Ca++ from INTRACELLULAR IP3-responsive stores

1)

2)

3)

Explanation / Answer

S.No Experimental strategy If calcium for extracellular stores If calcium from intracellular IP3 stores. 1 To culture cells in the absence of extracellular calcium. To check for intracellular calcium, if there is increase in calcium it is only due to IP3 Stores NA Yes 2 To culture cells in the presence of extracellular calcium with IP3 gene inhibitors NA Yes 3 To silence IP3 genes and to grow in the presence of calcium rich medium Yes NA

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