Flow Cytometry
Detection of Th17 (3-color) in Human Peripheral Blood PBMC
Source: Elabscience®Published: Dec 21,2023
|
Purpose |
Sample |
Antibody Collocation |
|
Adjust the voltage |
1 |
Blank |
|
Adjust compensation |
2 |
CD3-PerCP/Cyanine5.5 |
|
3 |
CD4-FITC |
|
|
4 |
IL-17A PE |
|
|
PE-FMO in combination with Isotype Control for auxiliary gating |
5 |
CD3-PerCP/Cyanine5.5, CD4-FITC; Mouse IgG1, κ Isotype Control-PE |
|
Full panel |
6 |
CD3-PerCP/Cyanine5.5, CD4-FITC, IL-17A- PE |
|
Marker |
Fluorochrome |
Clone No. |
Cat. No. |
|
CD3 |
PerCP/Cyanine5.5 |
OKT3 |
|
|
CD4 |
FITC |
SK3 |
|
|
IL-17A |
PE |
BL168 |
|
|
Mouse IgG1, κ Isotype Control |
PE |
MOPC-21 |
Tips:
1. After PBMC sorting, it is necessary to first use cytokine stimulating and blocking agents for stimulating and blocking culture (The reagents used in this experiment are: Cytokine Activation and Protein Blocking Kit (E-CK-A091). The cultivation conditions are as follows: after 1 hour of stimulation with a stimulating agent, add a blocking agent and incubate for another 4.5 hours), then collect cells for subsequent Flow Cytometry experiments.
2. PMA stimulation can cause partial endocytosis of CD4 on the surface of human T cells, so we need to choose the CD4 clone SK3 with minimal impact on endocytosis.
3. Isotype control for IL-17A is necessary, since the expression of cytokines is generally not high.
4. CD3+CD4+ IL-17A+ is Th17 type.
5. The Permeabilization buffer may cause significant damage to cells, so it is recommended that the cell precipitates formed after centrifugation should be dispersed into cell suspensions before adding the Permeabilization buffer to reduce cell damage.
