| KCl |
140 mm |
| HEPES |
10 mm |
| Na2ATP
|
0.5 mm |
| CaCl2 |
* mm |
| Ca2+ chelator
|
** 0.5 mm |
|
|
| Adjust the pH of the solution to 7.3 with 1 m KOH.
|
| * Use the online software Webmaxc Standard (http://www.stanford.edu/∼cpatton/webmaxcS.htm) to estimate the amount of CaCl2 that should be used to obtain the desirable free Ca2+ concentration ([Ca2+]f) in the final solution. Confirm the actual [Ca2+]f (<100 µm) in final solutions by Ca2+-sensitive dye fluorimetry. Do not use Ca2+ chelator for solutions with [Ca2+]f >100 µm. Calculate [Ca2+]f in these solutions by using the appropriate Ca2+ activity coefficients (Butler 1968; Vais et al. 2010).
|
| ** Use BAPTA (1,2-bis[o-aminophenoxy]ethane-N,N,N′,N′-tetraacetic acid) when 20 nm < [Ca2+]f ≤ 600 nm. Use 5,5′-dibromo BAPTA when 600 nm < [Ca2+]f ≤ 4 µm. Use HEDTA (hydroxyethylethylenediaminetriacetic acid) when 4 µm < [Ca2+]f ≤ 20 µm. Use NTA (nitrilotriacetic acid) when 20 µm < [Ca2+]f ≤ 100 µm. ATP contributes to Ca2+ buffering in solutions with [Ca2+]f >30 µm (Bers et al. 2010).
|
