MATERIALS AND METHODS
Lab cultured murine macrophages were cultured at 200,000 cells/well on a 96 well plate, in complete culture media (RPMI 1640, 10% FBS + 20 mM Hepes + 50μM 2-ME + 1% L-glutamine + 1% Penicillin/Streptomycin) at 37°C for two hours to allow for cell adhesion. Cells were then washed with media and treated.
There
are four levels of treatment:
1.
Media alone
2.
DMSO
3.
25µM inhibitor
4.
50µM inhibitor
Each of these levels was treated with six different stimuli:
1.
Media
2.
10ng/ml LPS
3.
2U/ml IFN
4.
LPS + IFN
5.
500 µg/ml
Echinacea
6.
Echinacea + IFN
DMSO (dimethyl
sulfoxide) is used as a negative control because it has been proven to be toxic
to macrophage at high concentrations.
LPS (lipopolysaccharide)
is a component of the cell wall of gram-negative bacteria and is a potent
macrophage stimulant and is used as a positive control in this experiment.
IFN
(Interferon gamma) is a cytokine produced by T cells and Natural Killer cells,
which activates macrophages to improve their phagocytic and killing abilities of
ingested pathogens and tumor cells.
ERK
Inhibitor (PD98059) has been shown to inhibit the phosphorylat8on and subsequent
activation of ERK preventing it from initiating activation of transcription
factor headed for cytokine expression.
Echinacea
polysaccharide is a laboratory preparation from the purple cone flower.
After two
hours the cells were washed three times using 200µl media/well with an
octapette removing all non-adherent cells. 100µl of the appropriate treatment
(level) was added to each well. After 30 minutes the appropriate 100 µl of
stimulus was added to each well (see table). The plate was then sealed and
placed in incubator for 48 hours.
Treatments
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
A |
Media |
Media |
Media |
Media |
||||||||
|
B |
LPS |
LPS |
LPS |
LPS |
||||||||
|
C |
IFN |
IFN |
IFN |
IFN |
||||||||
|
D |
LPS
+ IFN |
LPS
+ IFN |
LPS
+ IFN |
LPS
+ IFN |
||||||||
|
E |
EP |
EP |
EP |
EP |
||||||||
|
F |
EP +
IFN |
EP +
IFN |
EP +
IFN |
EP +
IFN |
||||||||
|
G |
|
|
|
|
|
|
|
|
|
|
|
|
|
H |
|
|
|
|
|
|
|
|
|
|
|
|
Levels of
treatment:
Column
1-3: Media
Column
4-6: DMSO
Column
7-9: 25µM inhibitor
Column 10-12: 50µM inhibitor
After 48
hours the supernatants collected from each triplicate were pooled in a 1.5 ml
tube and frozen at –20ºC until ready for further analysis.
ELISA
(Enzyme-linked Immunosorbent Assay)
A 96 well
plate was coated with 100µl of anti IL-6 antibody in each well overnight at 4ºC.
The plate was then washed three times with Wash Buffer. The plates were then
blocked with 200µl of Assay Diluent and left to incubate at room temperature
for 1 hour. During this time the standards were made by diluting the standard
stock solution supplied in the ELISA kit to 4000pg/ml, 2000pg/ml, 1000pg/ml,
500pg/ml, 250pg/ml, 125pg/ml and 62.5pg/ml using doubling dilutions. The plate
was then washed again three times and 100µl of standard or sample was added in
triplicate. The plate was then sealed and incubated at room temperature for 2
hours. After the incubation the plate was washed five times and 100µl of
working detector was added to each well. Again, the plate was sealed and
incubated at room temperature for an hour. The plate was washed seven times and
then 100µl of the Substrate Solution was added to each well in a darkened room.
After 30 minutes the reaction was stopped with 50µl of Stop Solution. The plate
was then analyzed with a microreader at a wavelength of 450 nm.
Materials
for ELISA:
Capture Antibody: anti-mouse IL-6 antibody
Wash
Buffer: Phosphate Buffer Saline/ Tween 20 Solution (0.05%)
Assay
Diluent: 10% Fetal Bovine Serum (FCS) in Phosphate Buffer Saline (PBS)
Standards:
for mouse IL-6
Working
Detector: Biotinylated Antibody +
Horseradish peroxidase
Substrate
Solution: TMB reagent
Stop
Solution: phosphoric acid