|
Purpose |
Sample |
Antibody Collocation |
|
|
Adjust the voltage |
1 |
Blank |
|
|
Adjust compensation |
2 |
CD3-PerCP/Cyanine5.5 |
|
|
3 |
CD4-FITC |
||
|
4 |
IFN-γ-PE |
||
|
5 |
IL-4-APC |
||
|
APC-FMO in combination with Isotype Control for auxiliary gating |
6 |
CD3-PerCP/Cyanine5.5、CD4-FITC、IFN-γ- PE、Rat IgG1, κ Isotype Control-APC |
|
|
PE-FMO in combination with Isotype Control for auxiliary gating |
7 |
CD3-PerCP/Cyanine5.5、CD4-FITC、IL-4-APC、Rat IgG1, κ Isotype Control -PE |
|
|
Full panel |
6 |
CD3-PerCP/Cyanine5.5, CD4-FITC, IFN-γ-PE, IL-4-APC |
|
Marker |
Fluorochrome |
Clone No. |
Cat. No. |
|
CD3 |
PerCP/Cyanine5.5 |
17A2 |
|
|
CD4 |
FITC |
RM4-5 |
E-AB-F1353C |
|
IFN-γ |
APC |
XMG1.2 |
|
|
IL-4 |
PE |
11B11 |
|
|
Mouse IgG1, κ Isotype Control |
APC |
HRPN |
|
|
Rat IgG1, κ Isotype Control |
PE |
HRPN |
E-AB-F09822E |
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Tips:
1. After the preparation and counting of the splenic single-cell suspension are completed, it is necessary to perform stimulation and inhibition culture using cytokine stimulation inhibitors (for experimental conditions of stimulation and inhibition, please refer to the instructions of the reagent kit used).
2. PMA stimulation can induce partial internalization of CD4 on the surface of human T cells. Therefore, it is necessary to select the CD4 clone RM4-5, which has minimal impact on internalization.
3. Isotype controls are required for IFN-γ and IL-4 because cytokine expression levels are generally low.
4. Th1 cells are characterized by the CD3⁺CD4⁺IFN-γ⁺ phenotype, while Th2 cells exhibit the CD3⁺CD4⁺IL-4⁺ phenotype.
5. Permeabilization reagents can cause significant damage to cells. It is recommended to first resuspend the cell pellet into a single-cell suspension after centrifugation before adding the permeabilization reagent, in order to minimize cell loss.
