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CBA Human Th1/Th2/Th17 cytokine kit (before you begin (steps) (1. prepare…
CBA Human Th1/Th2/Th17 cytokine kit
general info
purpose
measure (pg/ml)
IL-2
IL-4
IL-6
IL-10
TNF
INFα
IL-17A
priciple (sandwich)
capture beads with Ab
sample
PE-conjugated Ab
Advantage
broad dynamic range
less time
less sample volume
kit contents (80 tests)
A1-A7: 7 capture beads
B: PE dection Ab
C: cytokine standards
D: cytometer setup beads
set PMT voltage
compensation
E: PE/FITC positive control
F: wash buffer
G: assay diluent
H: serum enhancement buffer
goal
determine if T cell differentiation is being polirized to a Th1/Th2/Th17 response
Th17
differentiation: IL-6
secretion: IL-17A, TNF
multiple T cell differentiation pathway
proliferation: IL-2
regulation: IL-10
Th2
differentiation: IL-4
secretion: IL-4, IL-6
Th1
differentiation: IFN-g
secretion: IFN-g, TNF
before you begin (steps)
1. prepare standards (15 min)
2. mix capture beads (15 min)
3. dilute sample
4. prepare cytometer setup beads (15min) perform instrument setup
, perform during step 5
5. perform CBA cytokine assay (3h)
6. acauqire samples
7. data analysis
preparation
1. preparating standard
tube 1-10
dilution: 1:256 - 1:2 (2-9)
concentration: 20 - 5000 pg/ml (2-9)
2. mixing capture beads
10 µl capture beads/asay tube
3. diluting samples
procedure
5. performing CBA cytokine assay
50µl capture beads
50µl standard dilutions or sample
50µl PE detection Ab
incubate 3h (cytometer setup)
1ml wash buffer, centrifuge 100g, 5min
remove sup, add 300µl wash buffer, resuspend
7. data analysis