BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光,室温,-20°C储存,避免反复冻融。常规运输,低温运输,超低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
本产品用于检测人抗凝血中，Th17 细胞在T淋巴细胞 (或CD4⁺辅助T细胞) 中的比例。通常所说的Th1、Th2 和 Th17，是指在各种生理与病理条件下有能力分化为 Th1、Th2 和 Th17 的T辅助细胞 (严格意义上为 Th0 细胞)。静息状态 (即未受任何刺激，如人的正常生理状态) 下，Th0 分化为 Th1、Th2 和 Th17 的能力极弱，所以外周血中仅含有极少量的 Th1、Th2 和 Th17 细胞，这时所能检测到的 IFN-γ、IL-4 和 IL-17A 也微乎其微。而当 Th 细胞受到外界因素 (如刺激素、病原体等) 刺激，其中 Th0 即会向 Th1、Th2 或 Th17 分化，具体分化趋向取决于细胞因子的种类。此时，检测到的 IFN-γ、IL-4 或 IL-17A 也较多。实验中检测的 Th1、Th2 和 Th17 实际上是检测 Th 细胞对刺激素刺激的反应能力。
通常选用的刺激素为 Phorbol 12-Myristate 13-Acetate (PMA, 佛波酯) 和 Ionomycin (离子霉素)。其中 PMA 为 PKC (蛋白激酶C) 的激活物，PKC 则可激活下游众多的蛋白激酶的磷酸化，形成级联反应，导致许多蛋白的表达，进而引起T细胞的活化。在细胞内，PKC可被 DAG (二脂酰甘油) 和 Ca²⁺ 的共同作用而激活，因此在 Ionomycin (Ca²⁺的转运剂，可将细胞器内的 Ca²⁺ 转运至胞浆内) 的参与下，T细胞内PKC 可被进一步激活。可见，PMA与 Ionomycin 协同活化T细胞。
活化的T细胞可分泌多种细胞因子至细胞外，而流式细胞仪仅能检测细胞内的抗原，所以应将细胞因子阻断在胞内。细胞因子在高尔基体中合成，某些蛋白通过内质网运输以可溶性形式分泌至细胞外。破坏高尔基体即可切断细胞因子的转运途径，阻断其分泌。通常选用的阻断剂为 Brefeldin A (BFA, 布雷非德菌素 A) 和/或 Monensin (莫能霉素)。
此外，实验如何正确地设门以选定 Th 细胞是很重要的。Th 细胞的表面标志为 CD3⁺CD4⁺，而当用PMA 刺激人抗凝血 4 小时，会发现 CD4⁺ 的细胞明显减少，甚至消失，这是因为 PMA 会诱发细胞表面 CD4 分子被内吞。所以通常的做法是用 CD3 和 CD8 反设 CD4 细胞，即 CD3⁺CD8⁻的细胞被认为是 CD3⁺CD4⁺的细胞。
抗凝剂的选择也非常重要。使用肝素钠和肝素锂作为抗凝剂，不影响 Th 细胞的活化和细胞因子的分泌，而使用 EDTA 和枸橼酸钠作为抗凝剂则无法检测到细胞因子。此时，应使用PBMCs而不是抗凝血进行刺激。
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自备仪器和试剂：
1. 自备试剂
⁕ 细胞培养基（如RPMI1640、DMEM）
⁕ 胎牛血清（可选，根据实验需求准备）
⁕ 补偿调节微球（可选，根据实验需求准备）
⁕ 淋巴细胞分离液（可选，根据实验需求准备）
⁕ 4%多聚甲醛（可选）
2. 自备仪器
⁕ 12×75mm圆底流式管
⁕ 震荡器
⁕ CO₂恒温培养箱
⁕ 水平离心机（配备15ml离心管转子）
⁕ 流式细胞仪
实验步骤：
1. 样本收集与处理：
使用肝素或其它抗凝剂 (如 EDTA、枸橼酸钠) 作为抗凝剂，收集适量抗凝血，室温或2-8℃保存，并于当天进行检测。
2. 样本制备：
① 对于肝素抗凝血，取 125μl 抗凝血至流式管中，加入 125μl 不含血清的培养基和 1μl 佛波酯/离子霉素混合物 (250×) 和 1μl 布雷非德菌素A/莫能霉素混合物 (250×)。取 125μl 抗凝血和 125μl 不含血清的培养基，作为对照。混匀，37℃ 孵育 4-6 小时，每隔 1-2 小时取出震荡混匀。
② 对于使用其它抗凝剂 (如EDTA、枸橼酸钠) 抗凝血，用淋巴细胞分离液分离外周血单个核细胞 (PBMCs)。用含10%胎牛血清的培养基重悬沉淀，使细胞浓度为1×10⁷/ml。取 250μl PBMCs 至流式管中，加入 1μl 佛波酯/离子霉素混合物 (250×) 和 1μl 布雷非德菌素A/莫能霉素混合物 (250×)。以只含 PBMCs 的样本作为对照。混匀，37℃孵育 4-6 小时，每隔 1-2 小时取出震荡混匀。
注：佛波酯/离子霉素混合物 (250×) 和布雷非德菌素A/莫能霉素混合物 (250×) 极易挥发，使用后请及时旋紧管盖。
3. 从样本管和对照管中取 100μl 细胞悬液至新的流式管中，加入 5μl 抗人CD3, FITC 和 5μl 抗人CD8α, PerCP-Cy5.5。震荡混匀，室温避光孵育15 分钟。
4. 每管加入100μl 固定破膜剂试剂 A，震荡混匀，室温避光孵育15分钟。
5. 每管加入 1ml 预冷流式染色缓冲液 (1×)，300 x g 离心5分钟，弃上清。
注：液体尽量倒干净，不要有残留。
6. 每管加入100μl 固定破膜剂试剂 B 和 5μl 抗人 IL-17A, PE。震荡混匀，室温避光孵育15分钟。
7. 每管加入1ml 流式染色缓冲液 (1×)，300 x g 离心5分钟，弃上清。
8. 每管加入 500μl 流式染色缓冲液 (1×) 重悬，上机检测：或者加入 500μl 1-4%多聚甲醛重悬，2.8℃避光，于 24小时内检测。
流式检测：
1. 调节电压和补偿
使用补偿微球对流式细胞仪进行电压和补偿调节。补偿微球通常比目的细胞小，在调节 FSC/SSC 电压和细胞设门时需要注意。
2. 正确设门以得到 Th7 细胞在 CD3⁺ 或 CD4⁺辅助T细胞中的比例。
注：至少获取 20,000-30,000 CD3⁺CD8⁻T细胞。对于某一细胞因子，因人而异，分泌该细胞因子的细胞比例差异很大。为了进行统计学差异比较，请获取足够多的细胞样本。
荧光素激发波长和发射波长：
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注意事项：
1. 本产品仅用于科学研究，非诊断试剂，严禁用于临床诊断。
2. 请在产品标记的有效期内使用，过期试剂请勿使用。
3. 本产品各组件试剂不可与其他批号、其他来源的试剂混合使用，以免影响实验结果。
4. 佛波酯/离子霉素混合物 (250×) 和布雷非德菌素A/莫能霉素混合物 (250×) 极易挥发，使用后需立即旋紧管盖，避免试剂损耗和污染。
5. 所有孵育步骤需严格遵循避光要求，避免荧光素淬灭，影响检测信号。
6. 离心操作时，严格控制离心转速和时间，避免细胞破裂或丢失；弃去上清时，动作轻柔，避免残留液体影响后续染色。
7. 样本需在收集当天进行处理和检测，脾脏组织需保证新鲜，避免细胞死亡影响实验结果；经多聚甲醛固定的样本，需在24小时内完成检测。
8. 试剂储存需严格遵循推荐条件，避免冻融循环 (尤其是-20℃保存的试剂)，防止试剂失效。
特别说明：
发货时各组分依据储存温度 (室温/2-8℃/-20℃) 独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。
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.
危险品化学品经营许可证（带存储）