Bcecf am

Internal (cytoplasmic) pH was measured using the membrane-permeant, pH-sensitive fluorescent probe 2,7-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (BCECF-AM, Life Technologies) as described previously ^{30 (link)}. Experiments were done with or without CBS at 500 µg/mL. Experiments were also completed at 40 µg/mL and 80 µg/mL CBS with the same results (data not shown). Experiments were initiated at pH 7.4, then after about 175 seconds, HCl was added to drop the pH to 4.5.
Each experiment was calibrated independently. Once the fluorescence of the internal BCECF had been measured, 150 nM of the protonophore 3,3,4,5-tetrachlorosalicyanilide (TCS) was added to equilibrate the internal pH with that of the medium. HCl was then added to obtain minimum fluorescence of the dye, followed by the addition of NaOH to obtain maximum fluorescence of the dye. The internal pH was calculated using the equation: $${\text{pH}}_{\mathrm{i}}={\text{pK}}_{\mathrm{a}}+\text{log}(((\mathrm{R}-{\mathrm{R}}_{\mathrm{A}})/({\mathrm{R}}_{\mathrm{B}}-\mathrm{R}))\times ({\mathrm{F}}_{\mathrm{A}}(\mathrm{\lambda }2)/{\mathrm{F}}_{\mathrm{B}}(\mathrm{\lambda }2)))$$ where pK_a = pK_a of BCECF = 6.98, R = value at 502 nm/value at 436 nm for each data point, R_A = ratio at minimum fluorescence, R_B = ratio at maximum fluorescence, F_A(λ2) = minimum fluorescence at 436 nm, and F_B(λ2) = maximum fluorescence at 436 nm.

V-bottom well binding assay was performed as we previously described
[3 (link)]. Briefly, CHO-K1 cells WT, or
stably transfected with human ICAM-1 or mouse ICAM-1 were stained with
2’,7’-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein,
acetoxymethyl ester (BCECF-AM) (Life Technologies; Chelmsford, MA, USA).
V-bottom wells were coated with 5 µg/mL of human (or mouse) LFA-1 or
Mac-1 (R&D Systems; Minneapolis, MN, USA). Some of the wells were then
co-incubated with fibrinogen. BCECF-AM stained cells were incubated in V-bottom
wells with 1 mM Mg²⁺/Ca²⁺, or 1 mM
Mn²⁺/Ca²⁺ at 37 °C for 30 min. Then plates
were centrifuged at 200 × g for 5 min. Cells that did
not bind to plated ligand were accumulated at the center of the well.
Fluorescence was read with excitation 485 nm and emission 538 nm. The binding
% was defined as [(fluorescence intensity of CHO-K1 WT cell
samples)-(fluorescence intensity of CHO-K1 human (or mouse) ICAM-1 stable cell
samples)]/(fluorescence intensity of CHO-K1 WT cell samples) × 100
(%).

V-bottom plates were coated with 2 μg ICAM-1/ml (R&D systems, Minneapolis, MN) using standard protocols. HEK cells transfected with Mac-1 were stained with 2′,7′-bis-(2-carboxyethyl)-5- (and 6-)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Life Technologies) and incubated in HBS containing 10 mM EDTA, 1 mM MgCl₂/CaCl₂ or 1 mM MnCl₂ with or without isoflurane or sevoflurane at indicated concentrations at 37°C for 30 min. Following incubation, the plates were centrifuged at 200 × g for 5 min. Cells that did not bind to ICAM-1 were accumulated at the center of the well. Fluorescence was read at excitation = 485 nm and emission = 538 nm to assess HEK cell binding to ICAM-1. ICAM-1 binding percentage was defined as [(fluorescence intensity of EDTA sample) − (fluorescence intensity of samples)]/[fluorescence intensity of EDTA sample] × 100 (%). In some experiments, 5 μg/ml of LFA-1 or Mac-1 protein (a generous gift from Dr Timothy Springer [Boston Children's Hospital]) was coated on the 96 well V-bottom plates and HEK cells transiently transfected with ICAM-1 were used.