QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Christensen, K. A. Articles by Swanson, J. A. Search for Related Content PubMed PubMed Citation Articles by Christensen, K. A. Articles by Swanson, J. A. Social Bookmarking                         What's this?

pH-dependent regulation of lysosomal calcium in macrophages

¹ Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA
² The Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA

View larger version (9K): [in a new window]   Fig. 1. Plot of measured calcium Kd as a function of pH for the fluorescent calcium probes furaDx () and OGBDx ([UNK]). The solid line represents the predicted Kd of BAPTA using the constants measured by Tsien (Tsien, 1980) and the methods of Bers et. al. (Bers et al., 1994) to correct for temperature, ionic strength and pH. All measurements were performed at 22°C with constant ionic strength (0.130 M) and variable pH (4-7.2) in Hepes/MES pH buffers containing 1 mM Mg2+.

View larger version (15K): [in a new window]   Fig. 2. [Ca2+]lys and lysosomal pH: resting levels and effects of variable extracellular calcium and bafilomycin A1. (A) [Ca2+]lys. Black bars represent ratiometric measurements using a probe cocktail consisting of furaDx, FDx, and OGDx. The gray bar represents calcium measurements using fluorescence lifetime imaging with the calcium indicator OGBDx. The fluorescence ratio of furaDx was Rmin for cells incubated in 10 mM EGTA, therefore [Ca2+]lys was below the detection limit for furaDx at pH 4 (the bar represents the detection limit of furaDx at pH 4). Error bars represent s.e.m. (n10 cells). (B) Lysosomal pH. Error bars represent s.e.m. (n10 cells).

View larger version (16K): [in a new window]   Fig. 3. A time course showing ratiometric measurements of lysosomal pH () and [Ca2+]lys ([UNK]) following the addition of buffer containing 500 nM bafilomycin A1 (BAF). Lysosomes were labeled with a probe cocktail containing furaDx, FDx, and OGDx. Arrow indicates time of BAF addition. Error bars represent the s.e.m. (n9 cells).

View larger version (18K): [in a new window]   Fig. 4. A time course showing ratiometric measurements of lysosomal pH () and [Ca2+]lys ([UNK]) following the addition of buffer containing 10 mM NH4Cl. Lysosomes were labeled with a probe cocktail containing furaDx, FDx and OGDx. Arrow `a' indicates time of addition of NH4Cl. Arrow `b' indicates time of addition of NH4Cl-free buffer (RB) causing rapid reacidification and restoration of high [Ca2+]lys. Error bars represent the s.e.m. (n9 cells).

View larger version (22K): [in a new window]   Fig. 5. Data from a time-course experiment showing the effects of NH4Cl on macrophage [Ca2+]cyt and [Ca2+]lys. (A) [Ca2+]cyt in activated macrophages ([UNK]) following addition of NH4Cl and control cells (; activated macrophages without addition of NH4Cl). (B) [Ca2+]lys ([UNK]) and lysosomal pH () in activated macrophages following addition of NH4Cl. (C) [Ca2+]lys ([UNK]) and lysosomal pH () in control cells (no NH4Cl added). To measure [Ca2+]cyt, cells were loaded with FFP-18AM. To measure [Ca2+]lys, cells were loaded with a probe cocktail consisting of furaDx, FDx, and OGDx. Arrows show times of addition of ammonium chloride. Error bars represent s.e.m. (n=30 cells for [Ca2+]cyt measurements and n15 cells for [Ca2+]lys measurements).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter