This product is designed to determine the proportions of Th17 cells within T‑lymphocytes (or CD4⁺ helper T‑cells) in human anticoagulated whole blood. The commonly‑referred Th1, Th2 and Th17 denote helper T‑cells (strictly speaking, Th0 cells) that possess the capacity to differentiate into Th1, Th2 and Th17 under diverse physiological and pathological conditions. Under quiescent conditions (i.e., in the absence of any stimulation, such as the normal physiological state in humans), Th0 cells exhibit very poor differentiation potential toward Th1, Th2 and Th17. Consequently, only trace amounts of Th1, Th2 and Th17 cells are present in peripheral blood, and correspondingly minimal detectable levels of IFN‑γ, IL‑4 and IL‑17A. Upon stimulation of helper T‑cells by external factors (e.g., stimulants, pathogens), Th0 cells differentiate into Th1, Th2 or Th17 subsets. The specific differentiation bias is dictated by the cytokine milieu, at which point abundant IFN‑γ, IL‑4 or IL‑17A can be detected. In experimental settings, the readout for Th1, Th2 and Th17 functionally reflects the responsiveness of helper T‑cells to stimulant challenge.
Phorbol 12‑Myristate 13‑Acetate (PMA) and Ionomycin are the stimulants of choice. PMA is an activator of Protein Kinase C (PKC). Activated PKC triggers phosphorylation of numerous downstream protein kinases, initiating a cascade reaction that drives the expression of multiple proteins and ultimately T‑cell activation. Intracellularly, PKC is activated synergistically by diacylglycerol (DAG) and Ca²⁺. Accordingly, in the presence of Ionomycin (a Ca²⁺ ionophore that shuttles Ca²⁺ from intracellular organelles into the cytosol), intracellular PKC in T‑cells undergoes further activation. Hence, PMA and Ionomycin exert synergistic effects to activate T‑cells.
Activated T‑cells secrete a broad spectrum of cytokines into the extracellular space. Since flow cytometry can only detect intracellular antigens, cytokines must be retained inside the cells. Cytokines are synthesized in the Golgi apparatus; some proteins are transported via the endoplasmic reticulum and secreted extracellularly as soluble molecules. Disruption of Golgi function blocks cytokine trafficking and subsequent secretion. Brefeldin A (BFA) and/or Monensin are routinely used as secretion inhibitors.
In addition, a proper gating strategy for identifying helper T‑cells is critical for the assay. Helper T‑cells are phenotypically defined as CD3⁺CD4⁺. Upon 4‑hour PMA stimulation of human anticoagulated whole blood, CD4⁺ cells are markedly decreased or even undetectable. This is because PMA induces internalization of surface CD4 molecules. Accordingly, a common indirect‑gating approach is applied: CD3⁺CD8⁻ cells are considered equivalent to CD3⁺CD4⁺ cells.
Anticoagulant selection is also critical. Sodium heparin and lithium heparin do not interfere with helper T‑cell activation or cytokine secretion. By contrast, no cytokines can be detected when EDTA or sodium citrate is used as the anticoagulant. Under these conditions, PBMCs, rather than anticoagulated whole blood, should be used for stimulation.
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Required User‑Supplied Instruments and Reagents:
1. User‑Supplied Reagents
⁕ Cell culture medium (e.g., RPMI 1640, DMEM)
⁕ Fetal bovine serum (optional, prepare according to experimental requirements)
⁕ Compensation beads (optional, prepare according to experimental requirements)
⁕ Lymphocyte separation medium (optional, prepare according to experimental requirements)
⁕ 4% paraformaldehyde (optional)
2. User‑Supplied Instruments
⁕ 12×75 mm round‑bottom flow cytometry tubes
⁕ Vortex mixer
⁕ CO₂ incubator
⁕ Horizontal centrifuge (with rotor compatible with 15 mL centrifuge tubes)
⁕ Flow cytometer
Experimental Procedure:
1. Sample Collection and Processing
Collect an appropriate volume of anticoagulated blood using heparin or other anticoagulants (e.g., EDTA, sodium citrate). Store samples at room temperature or 2‑8 °C and perform detection on the same day.
2. Sample Preparation
① For heparin‑anticoagulated blood: Transfer 125 μL anticoagulated blood into a flow cytometry tube. Add 125 μL serum‑free medium, 1 μL PMA/Ionomycin Mixture (250×), and 1 μL BFA/Monensin Mixture (250×). For the control, combine 125 μL anticoagulated blood with 125 μL serum‑free medium. Mix thoroughly and incubate at 37 °C for 4‑6 hours. Vortex the tubes every 1‑2 hours during incubation.
② For blood collected with other anticoagulants (e.g., EDTA, sodium citrate): Isolate peripheral blood mononuclear cells (PBMCs) using lymphocyte separation medium. Resuspend the cell pellet in medium supplemented with 10% fetal bovine serum to a cell concentration of 1 × 10⁷ cells/mL. Transfer 250 μL PBMC suspension into a flow cytometry tube, then add 1 μL PMA/Ionomycin Mixture (250×) and 1 μL BFA/Monensin Mixture (250×). Use PBMCs without added stimulants as the control. Mix well and incubate at 37 °C for 4‑6 hours. Vortex every 1‑2 hours.
Note: PMA/Ionomycin Mixture (250×) and BFA/Monensin Mixture (250×) are highly volatile. Tighten tube caps promptly after use.
3. Transfer 100 μL cell suspension from sample and control tubes into new flow cytometry tubes. Add 5 μL Anti‑Human CD3, FITC and 5 μL Anti‑Human CD8α, PerCP‑Cy5.5. Vortex and incubate for 15 minutes at room temperature protected from light.
4. Add 100 μL FIX & PERM Medium A to each tube. Vortex and incubate 15 minutes at room temperature in the dark.
5. Add 1 mL pre‑chilled Flow Cytometry Staining Buffer (1×) to each tube. Centrifuge at 300 × g for 5 minutes and discard supernatant.
Note: Remove supernatant completely and avoid residual liquid.
6. Add 100 μL FIX & PERM Medium B, and 5 μL Anti-Human IL-17A, PE per tube. Vortex and incubate 15 minutes at room temperature, protected from light.
7. Add 1 mL Flow Cytometry Staining Buffer (1×) to each tube. Centrifuge at 300 × g for 5 min and discard supernatant.
8. Resuspend cells in 500 μL Flow Cytometry Staining Buffer (1×) and acquire data on flow cytometer.
Alternative: Resuspend in 500 μL 1‑4% paraformaldehyde. Store at 2‑8 °C in dark and complete acquisition within 24 hours.
Flow Cytometry Acquisition:
1. Voltage and Compensation Setup
Use compensation beads to configure voltage and compensation parameters for the flow cytometer. Compensation beads are generally smaller than target cells; this point should be noted when adjusting FSC/SSC voltages and establishing cell‑gating strategies.
2. Perform proper gating to calculate the proportions of Th7 cells within CD3⁺ or CD4⁺ helper T‑cells.
Note: Acquire a minimum of 20 000‑30 000 CD3⁺CD8⁻ T‑cell events. The percentage of cytokine‑secreting cells varies greatly among individuals for any given cytokine. Collect sufficient cell events to enable valid statistical comparison.
Fluorophore Excitation and Emission Wavelengths:
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Precautions:
1. This product is for research‑use‑only (RUO). It is not a diagnostic reagent and must not be used for clinical diagnosis.
2. Use the product within the shelf‑life indicated on the label. Do not use expired reagents.
3. Do not mix kit components with reagents from alternative lot numbers or other sources, as this may compromise experimental results.
4. PMA/Ionomycin Mixture (250×) and BFA/Monensin Mixture (250×) are highly volatile. Immediately tighten tube caps after use to prevent reagent loss and contamination.
5. All incubation steps must be performed protected from light to prevent fluorophore photobleaching and signal loss.
6. Centrifuge using defined speed and duration to avoid cell lysis or cell loss. Decant supernatants gently; residual liquid may interfere with subsequent staining.
7. Process and test samples on the day of collection. Spleen tissue must be kept fresh to prevent cell death that could distort assay results. Paraformaldehyde‑fixed samples must be acquired within 24 hours.
8. Store all reagents under recommended conditions. Avoid repeated freeze‑thaw cycles (especially for reagents stored at −20 °C), which may cause reagent inactivation.
Important Notes：
Each component is individually packaged according to its storage temperature (Room temperature/2-8 °C/-20 °C) upon shipment, with differentiated temperature-controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf-life is 12 months from the date of manufacture.
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