Biology Reference
In-Depth Information
used: 8H4 (1:3000), 3F4 (1:5000), ferritin (1:1000), Tf (1:6000), TfR (1:3000), actin
(1:7500), secondary antibodies conjugated with horseradish peroxidase (1:6000). Im-
munoreactive bands were visualized by ECL (Amersham Biosciences Inc.).
Measurement of Intracellular Calcein-chelatable Iron
Cellular LIP was assayed as described by Tenopoulou et al. [43] using the iron sensi-
tive fluorescent dye calcein. When incubated with cells as a lipophilic calcein-AM-
ester (molecular probes), it enters the cells and is cleaved by cellular esterases to re-
lease calcein that binds iron and is quenched by this reaction. Upon addition of the cell
permeable iron chelator SIH, iron is released from calcein that regains its fluorescence
(recorded at λ
ex
488 nm and λ
em
518 nm). Briefly, 5 × 10
5
M17 cells or cell lines ex-
pressing PrP
C
and mutant PrP forms plated in 35 mm Petri dishes were washed with
PBS containing 1 mg/ml BSA and 20 mM Hepes, pH 7.3 and incubated with 0.25
μM calcein-AM for 20 min at 37°C in same buffer. After calcein loading, cells were
trypsinized, washed and re-suspended in 1.0 ml of the above buffer without calcein-
AM and placed in a 24 well micro-plate in a thermostatically controlled (37°C) fluo-
rescence plate reader (Microtek). The fluorescence was monitored at λ
ex
488 nm and
λ
em
518 nm. Iron-induced quenching of calcein was reduced by the addition of 20 μM
SIH. Cell number and viability was checked by Trypan Blue dye exclusion and results
were expressed as ΔF/10
6
cells.
Detection of Iron with Ferene S
Cell lines cultured overnight in complete medium or in the presence of 0.1 mM FAC
were washed with PBS supplemented with EDTA to chelate surface bound iron and
pelleted. The pellet was dissolved in 50 μl of acetic acid and equal amount of protein
(50 μg) was spotted on a PVDF membrane and immersed in a freshly prepared solu-
tion of Ferene S (0.75 mM 3-(2-pyridyl)-5, 6-bis(2-(-furyl sulfonic acid)-2, 4-triazine,
2% (v/v) acetic acid, 0.1% thioglycolic acid) (24) for 30 minutes at 37°C. Ferene
reacts with iron in the presence of acetic acid and thioglycolic acid to form a dark blue
complex. Stained membranes were de-stained with 2% acetic acid and scanned.
Stimulation of Endocytosis with 3F4 Antibody
The M17 and PrPC cells were cultured in DMEM supplemented with 5% FBS and
1% PSF at 37°C in a humidified atmosphere in absence or presence of 1 μg/ml of 3F4
for 5 days [26], [27]. Medium containing 3F4 was replaced every second day and care
was taken to make sure that the cells did not achieve confluency. On the fifth day, cells
were washed and incubated with serum free DMEM for 1 hr, followed by radiolabel-
ing with
59
FeCl
3
-citrate complex in DMEM for 4 hr as above. In a separate experimen-
tal paradigm, N2a, M17, and PrP
C
cells were radiolabeled as above in the presence of
12 μg/ml of 3F4 or Thy-1 4 hr. After labeling, cells were washed, lysed in native lysis
buffer, and analyzed as above.
Immunostaining and Fluorescence Microscopy
Cell lines subjected to different experimental conditions were processed for immunos-
taining as described in a previous report [41].
