desired buffer. The compound addition is
200 µL/well /96 well plate
7. Run the pH assay by monitoring the fluorescence
at λex = 490/λem = 535 nm (cut off at 515 nm) or
λex = 505/λem = 535 nm
λEx/λEm = 470/550 nm with cutoff
at 515 nm.
Results
1. The reading (RFU) obtained from the blank
standard well is used as a negative control.
2. Subtract the
filter that allows passage
of all wavelengths longer than 515 nm will result in a yellow-green emission color, where as a notch filter, (eg. 515-565 nm) will result in a green
emission color. Most filters
volume (in milliliters) of the assay
12.6 = Millimolar extinction coefficient of 4-nitrocatechol at 515 nm.
0.1 = Volume (in milliliter) of enzyme used
and LC FastStart DNA MasterPLUS SYBR Green I* (Cat. No. 03 515 869 001) according
to the procedures within the Instructions for Use.
Fig. 2: Linear control reaction:
Control reaction with PBGD primers
myeloid and
plasmacytoid dendritic cells to lymphoid organs and inflamed skin. J. Exp. Med. 201:509-
515.
Wittamer et al. (2003) Specific recruitment of antigen-presenting cells by chemerin, a novel
45 min,
+37°C
6 Washing: Wash cells twice in PBS 2× 2 min,
+15 to +25°C
7 Analysis: Analyze on a flow cytometer (488nm
excitation using a 515 nm bandpassfilter for
detection) or counterstain
does not affect enzyme acitivity.
1. Add up to 200 µg of glycoprotein to a
microcentrifuge tube. Bring the volume to 37.5 µL
with deionized water.
2. Add 10 µL of 5X Reaction Buffer (Catalog number
Consumables
Type Model Unit Cost Type Model Unit Cost
Single-use Mixer Bag MIX0100LB05 $515
Total: - Total: $515
Waste
Volume per Batch (m3) Cost ($/m3) Total Cost ($) Weight per Batch (kg) Cost
A. M., Mol. Cell Endocrinol., 49, 1-16,
(1987).
7. de Almeida, et al., J. Cell Sci., 107, 507-515 (1994).
8. Wilkie, T. M., and Yokoyama, K. S., Soc. Gen.
Physiol. Ser., 49, 249-270 (1994).
9
minutes at -20°C.
(iii). Wash the cells with PBS, centrifuging at 200 x g for IO minutes.
Fixation method B
(i). Incubate l-2 x 1 06 cells in 100% methanol for 30 minutes at -20°C.
(ii). Wash the
optimal working dilutions
by titration test.
References
1. Mori, Y. et al., Neuroreport, 9, 507-515 (1998).
2. Wes, P.D. et al., Proc. Natl. Acad. Sci. USA, 92,
9652-9656 (1995).
3. Harteneck, C., et
A. M., Mol. Cell Endocrinol., 49, 1-16,
(1987).
7. de Almeida et al., J. Cell Sci., 107, 507-515 (1994).
8. Wilkie, T. M., and Yokoyama, K. S., Soc. Gen.
Physiol. Ser., 49, 249-270 (1994).
ANTIBODY: Incubate the sections with a 1:50 to 1:200 dilution of goat anti-rabbit in Solution D
containing 1% non-specific serum for either 3 hrs at 20°C or 2 hr at 37°C. Then wash the sections, 3 times