Part 2: CW/WS

Question 1

3 examples of fluorescent calcium sensitive indicators

Answer 1

• Fura-2
• Indo-1
• Quin-2

Question 2

3 issues with using calcium-sensitive microelectrodes:

Answer 2

• Cell must withstand impalement
• Slow response time
• Difficulty in calibration

Question 3

3 issues with fluorescent calcium-sensitive indicators:

Answer 3

• Loading difficulties
• Indicator loss/sequestration
• High buffer capacity

Question 4

Components of recombinant aequorin:

Answer 4

• Two distinct units (apoaequorin, apoprotein)
• Prosthetic group: coelenterazine (added to reconstitute active aequorin protein) -> calcium measurements via light emission

Question 5

What two methods are used for measuring aequorin luminescence?

Answer 5

• Luminescence imaging (complex and expensive)
• Luminometry (using photon counting tube; cheaper)

Question 6

What two ways can aequorin-derived calcium measurements be analysed?

Answer 6

• As relative luminescence values in arbitrary units
• Calibrated to give concentration using empirically derived equations

Question 7

What parameters are typically used in analysis of aequorin data?

Answer 7

• Total change in cytosolic free calcium
• Magnitude and timing of phase(s)
• Mean +/- SEM should be considered in all cases