Bio-Rad Bio-Plex Pro™ Rat Cytokine, Chemokine, and Growth Factor Assays - Manual (Page 20)

18
Lysates
The Bio-Plex cell lysis kit is required for lysate preparation (available
separately, catalog #171-304011 and #171-304012). Refer to bulletin #5297
for a list of published articles on cytokine analysis in tissue samples.
1. Prepare the cell or tissue lysates according to the instructions
provided with the Bio-Plex cell lysis kit. The protease inhibitors factor I
and factor II are included in the kit. PMSF needs to be added to lysis
buffer at a final concentration of 2 mM. The lysates should be free of
particulate matter.
2. Determine the total protein concentration of the lysate. It may be
necessary to test lyse your samples with different volumes of lysing
solution to obtain the specified protein concentration range.
3. Dilute at least 1:2 in sample diluent + 0.5% BSA, to a final protein
concentration of 200–900 µg/ml. For analytes with high expression, a
lysate protein concentration as low as 50 μg/ml may be sufficient.
Note: For optimum antibody binding during sample incubation, it
is important to dilute lysates as much as possible to reduce the
detergent concentration.
4. If the lysate is not tested immediately, store at –20°C to -70°C. Avoid
repeated freeze-thaw cycles.
Sample Dilution for ICAM-1 and VCAM-1
ICAM-1 and VCAM-1 are typically found at high concentrations in the
blood; therefore, higher sample dilutions are required to achieve measurable
concentrations within the standard curve range.
For human ICAM-1 and VCAM-1 assays, dilute serum or plasma 1:100.
For example:
n
First dilution (1:4)—10 µl sample + 30 µl Bio-Plex sample diluent
n
Second dilution (1:25)—5 µl from the first dilution + 120 µl
Bio-Plex standard diluent