Download PDF

Fabienne Charrier-Savournin, PhD, Fanny Pleniere, Stephanie Douzon | Cisbio Bioassays
Caroline Cardonnel, PhD, Laurence Monnet | Field Applications Scientists | Molecular Devices

Introduction

Pro- and anti-inflammatory cytokines play a central role in autoimmunity and inflammatory and infectious diseases. They are also key players in metabolic disorders and oncology, particularly the anti-tumor immune response. Homogeneous Time Resolved Fluorescence (HTRF) from Cisbio Bioassays provides a versatile platform for the quantitation of various cytokines and chemokines. HTRF is perfectly suited to monitoring cytokine and chemokine release in cell-based experiments, especially physiological models such as peripheral blood mononuclear cells (PBMC). Typically, the modulation of cytokine release relies on the use of pharmacological compounds either to induce or inhibit their secretion. To gain further insight into the mechanism of action of such drugs, it is necessary to carefully analyze their effects, particularly the effect on cell viability.

This application note demonstrates how to perform a relevant analysis of cytokine secretion along with cell viability, using the SpectraMax i3x^® Multi-Mode Microplate Reader with SpectraMax^® Minimax^® 300 Imaging Cytometer. This allows users to measure HTRF and cell viability on a well-by-well or cell-by-cell basis, as well as confirming assay quality by monitoring cells’ appearance. We describe (i) how to induce cytokine secretion from treated PBMC, (ii) how to measure cytokines with HTRF, (iii) how to evaluate viability of PBMC using the EarlyTox™ Cell Integrity Kit from Molecular Devices, and (iv) how to normalize cytokine concentration to PBMC viability.

Materials and methods

Frozen human PBMC were thawed and seeded at 50,000 and 100,000 cells per well in a 96-well, black-wall, clear-bottom microplate in 200 µL of RPMI + 10% fetal calf serum (FCS). Prior to cell treatment, PBMC seeding was monitored by imaging with the MiniMax cytometer using the transmitted light channel.

Cell treatments

To modulate IL6, IL8, TNFα, and IL1β secretion, PBMC were cotreated for 16 hours with increasing concentrations of dexamethasone, a glucocorticoid known to down-regulate pro-inflammatory cytokine secretion, plus LPS at 0.2 µg/mL to induce cytokine secretion. To modulate IL2 and IFNγ secretion, PBMC were cotreated for 16 hours with increasing concentrations of dexamethasone, plus PMA (0.5 ng/mL) and ionomycin (1 µg/mL).

HTRF cytokine assays

After an overnight incubation, 16 µL of supernatant from each well was transferred to a 384-well, low-volume microplate (Greiner #784075) to perform HTRF cytokine assays. Briefly, each sample was used neat or diluted 1:10 or 1:20 to work within the linear range of each assay. Cytokine concentrations were interpolated from the corresponding standard curve, consisting of standards diluted in RPMI + 10% FCS (HTRF cytokine kits: 62HIFNGPEG, 62HIL02PEG, 62HIL1BPEG, 62HIL06PEG, 62HIL08PEG, 62HTNFAPEG).

The SpectraMax i3x reader was used to read HTRF cytokine assays with a preconfigured protocol in SoftMax^® Pro Software.

To view the complete workflow of how to normalize HTRF cytokine assays to cell viability, please visit moleculardevices.com/ htrf-workflow

EarlyTox Cell Integrity Assay

Cell viability was subsequently evaluated with the EarlyTox™ Cell Integrity Kit on the cells following supernatant aspiration. The EarlyTox Cell Integrity Kit (Molecular Devices, P/N: R8213) uses two DNA-binding dyes: cell-permeant Live Red Dye (labels the nuclei of all cells) and cellimpermeant Dead Green Dye (labels dead cells only). Cell viability was evaluated with the two-color fluorescence imaging capability of the Minimax cytometer (green: 460 nm ex/541 nm em, and red: 625 nm ex/713 nm em) and the cellular analysis features of SoftMax Pro Software.

Data analysis

Data were generated and analyzed using SoftMax Pro Software.

• HTRF ratio and data reduction were performed automatically in the preconfigured protocol following Cisbio guidelines. (http://www.cisbio.com/usa/drug-discovery/htrf-ratio-and-datareduction)
• Means and standard deviations (SD) are represented on the graphs.
• Normalization procedure: Normalized [cytokine]pg/mL = [cytokine]pg/mL ÷ number of living cells

Results

Control of PBMC seeding prior to cell treatments

Prior to cell treatment, human PBMC were imaged in the transmitted light channel to confirm proper seeding. These cells have a diameter of less than 6 µm and as a result can be difficult to count individually in a transmitted light image. Therefore, we implemented a user-defined custom analysis to calculate cell density as a percent of covered area per image. This analysis calculated 19.7% covered area representing 50,000 PBMC per well and 35.9% covered area for 100,000 cells per wells, which corresponds to a ratio of 1.82 (data not shown). These results confirm that this new custom data analysis successfully identified objects in field analysis and enabled an accurate percentage area coverage per well.

Modulation of cytokine release induced by LPS or PMA/ionomycin after dexamethasone treatment

As expected, LPS efficiently induced the secretion of IL1β, IL6, IL8 and TNFα (Figure 1) while co-stimulation with 5 ng/mL PMA + 1 µg/mL ionomycin induced a maximal release of IL2 and IFNγ.

Figure 1. Cytokine secretion upon LPS stimulation, measured by HTRF assays on the SpectraMax i3x reader.

Interestingly, both IL2 and IFNγ secretion were significantly decreased in the presence of 2 µg/mL of ionomycin (data not shown).

Dexamethasone caused a significant decrease in cytokine release induced by either LPS or PMA/ionomycin treatments (Figure 2).

Figure 2. Effect of dexamethasone on cytokine secretion. Dose-response curves for TNFα, IL2 and IFNγ not shown.

Effect of LPS, dexamethasone, and PMA/ionomycin treatment on cell viability monitored with the EarlyTox Cell Integrity Assay

The EarlyTox data analysis relied on cell count using the red channel (size and fluorescence intensity threshold) and on identification of live and dead cell populations. Using the Classification feature in SoftMax Pro Software, cells with nuclei labeled red only were classified as live, and cells with nuclei labeled both red and green were classified as dead. Identified objects were visualized in the software by red (live) and blue (dead) masks (Figures 3 and 4).

Figure 3. LPS + Dexamethasone treated PBMC. Cells were recognized as live (red masks) but no dead cells (blue masks) were identified using the Classification feature in SoftMax Pro Software.

Figure 4. PMA + ionomycin treated PBMC. Cells were identified as live (red masks) or dead (blue masks) in clusters using the Classification feature in SoftMax Pro Software.

LPS + dexamethasone treatment did not cause any noticeable effects either on cell viability or cell morphology compared to untreated cells (Figure 3). PMA and ionomycin stimulation treatment induced formation of cell clusters and 10-13% dead cells (Figure 4 and Table 1).

Table 1. Results of EarlyTox Cell Integrity Assay showing effect of LPS or PMA/ionomycin on PBMC cell viability (percentage of dead cells).

To gain further insight into PMA/ionomycin effects on cell viability and morphology, PBMC were treated with increasing doses of PMA and ionomycin separately. The same methodology as previously described was applied (images not shown). As reported in Figure 5, cell death significantly increased with increasing concentrations of the two compounds, indicating that both drugs mediate cytotoxicity. Moreover, our data showed that PMA is almost 2 times more cytotoxic than ionomycin (15% dead cells with 50 ng/mL PMA compared to 7% of dead cells at 2 µg/mL ionomycin).

Figure 5. Cytotoxic effect of PMA and ionomycin when added separately.

Normalization of cytokine production to cell viability

Integration of PBMC viability with cytokine secretion data represents the last stage of an accurate analysis of the biological response. To perform the normalization, the concentration of cytokine previously determined by HTRF was divided by the number of living cells, determined by the EarlyTox Cell Integrity Assay.

In Figure 6, the concentration of cytokine was expressed per number of living cells, and the pharmacological window and IC₅₀ were reported in Table 2. As expected, since dexamethasone did not induce any significant cytotoxic effect, the pharmacological results were comparable before and after normalization.

Figure 6. Effect of dexamethasone on normalized cytokine secretion. Dose-response curves for TNFα, IL2, and IFNγ are not shown.

Table 2. Pharmacological window (signal/background) and IC₅₀ values for stimulated PBMC.

However, normalizing PMA/ionomycinstimulated IL2 or IFNγ secretion to cell viability values revealed a concentrationdependent effect of PMA (Figure 7) that was not previously detected. This highlights that the normalization of cytokine secretion (or other biomarkers) to the number of live cells can reveal relevant biological responses.

Figure 7. Effect of different concentrations of PMA/Ionomycin on normalized cytokine secretion. Top, hIL2 secretion normalized to number of live cells. Bottom, hIFNγ secretion normalized to number of live cells.

Conclusion

This work clearly establishes the cytotoxic effect of PMA and ionomycin, in contrast to dexamethasone, which does not induce any noticeable cytotoxicity. Cell viability results obtained with the EarlyTox Cell Integrity Kit show a visible concentrationresponse effect of PMA or ionomycin.

Cisbio Bioassays offers an easy-to-use and sensitive method for evaluating cytokine secretion and determining the cytotoxic effect of drugs on cytokine release from suspension cells such as PBMCs. The combined capabilities of the SpectraMax i3x reader and MiniMax cytometer enable HTRF cytokine quantitation and cell viability assessment using the EarlyTox Cell Integrity Kit.

Fabienne Charrier-Savournin, PhD, Fanny Pleniere, Stephanie Douzon | Cisbio Bioassays
Caroline Cardonnel, PhD, Laurence Monnet | Field Applications Scientists | Molecular Devices

简介

已知促炎和抗炎细胞因子在自身免疫、炎 症调节和传染病中发挥着重要的作用。同 时在代谢紊乱、肿瘤学也起着重要作用， 尤其是抗肿瘤免疫应答。Cisbio 公司提供 各种细胞因子和趋化因子定量的均相时间 分辨荧光 (HTRF) 检测试剂盒。HTRF 技术 非常适合监测细胞因子和趋化因子在细胞 实验中的释放，特别是像外周血单个核细 胞 (PBMC) 这样的物理模型。通常，细胞 因子释放的调节依赖于使用药物化合物来 诱导或抑制它们的分泌。为了进一步了解 这些药物的作用机制，需要我们仔细分析 它们的作用，特别是对细胞活力的影响。 本应用介绍了如何使用 SpectraMax i3x 多 功能微孔板读板机和 SpectraMax Minimax 300 细胞成像仪如何对细胞因子分泌和细 胞活性进行分析。该方法可以逐孔/逐细 胞进行 HTRF 和细胞活性检测，并通过细 胞形态来确定其质量。本文将介绍 (i) 如何 经过处理的 PBMC 中诱导细胞因子分泌， (ii) 如何通过HTRF方法检测细胞因子，(iii) 如何用 Molecular Devices 公司的 EarlyTox 细胞完整性检测试剂盒评估 PBMC 活性， (iv) 何将细胞因子浓度转化成 PBMC 活性。

本应用介绍了如何使用 SpectraMax i3x 多 功能微孔板读板机和 SpectraMax Minimax 300 细胞成像仪如何对细胞因子分泌和细 胞活性进行分析。该方法可以逐孔/逐细 胞进行 HTRF 和细胞活性检测，并通过细 胞形态来确定其质量。本文将介绍 (i) 如何 经过处理的 PBMC 中诱导细胞因子分泌， (ii) 如何通过HTRF方法检测细胞因子，(iii) 如何用 Molecular Devices 公司的 EarlyTox 细胞完整性检测试剂盒评估 PBMC 活性， (iv) 何将细胞因子浓度转化成 PBMC 活性。

材料和方法

解冻冻存人类 PBMC，并将其以 50,000 和 100,000 细胞/孔接种于底透黑色的 96 孔 板中，每孔接种 200 µl RPMI + 10% 胚牛 血清 (FCS)。在细胞处理前，用 MiniMax 细 胞成像仪透射光检测 PBMC 接种量。

细胞处理

为了调节 IL6, IL8, TNFα 和 IL1β 分泌， PBMC 处理 16 小时，地塞米松浓度递增， 一个已知的糖皮质激素会抑制促炎细胞因 子分泌，再加入 0.2 μg/mL LPS 诱导细胞 因子分泌。为了调节 IL2 和 IFNγ 分泌， PBMC 处理 16 小时，地塞米松浓度递增， 再加入 0.5 ng/mL PMA 和 1 μg/mL 离子毒 素。

HTRF 细胞因子分析

过夜培养后，每孔上清 16 µl 转移至小体积 384 孔板 (Greiner #784075) 进行 HTRF 细 胞因子分析。每个样品用1∶10/1∶20 比例 稀释，这样每个分析都在其线性范围内。 细胞因子浓度根据对应的标准曲线计算， RPMI + 10% FCS 稀释成标准品 (HTRF细胞 因子试剂盒：62HIFNGPEG, 62HIL02PEG, 62HIL1BPEG, 62HIL06PEG, 62HIL08PEG, 62HTNFAPEG) 采用 SoftMax Pro 软件中预 设的模板在 SpectraMax i3x 微孔板读板机 上进行 HTRF 细胞因子分析。

EarlyTox 细胞完整性检测分析

采用 EarlyTox 细胞完整性检测试剂盒对细 胞上清进行细胞活性的评估。EarlyTox 细 胞完整性检测试剂盒 (Molecular Devices, P/N:R8213) 有 2 个 DNA 结合染料：活细胞 红色染料 ( 标记所有细胞的细胞核 ) 和死细 胞绿色染料 ( 只标记死细胞 ) 。细胞活性用 两种颜色的荧光染料在 MiniMax 细胞成像 仪上成像 ( 绿色：460 nm 激发/541 nm 发 射，红色：625 nm 激发/713 nm 发射 )， 用 SoftMax Pro 软件进行分析。

数据分析

SoftMax Pro 软件进行数据采集和分析

• HTRF 比值和数据处理都可以直接调用软 件中根据 Cisbio 试剂盒预设的程序自动 处 理 。 ( h t t p : / / w w w . c i s b i o . c o m / usa/drug-discovery/htrf-ratio-and-data-reduction)
• 平均值和标准偏差 (SD) 会在图中显示
• 归一化过程：归一化【细胞因子】 pg/mL =【细胞因子】pg/mL ÷ 活细胞 数量

结果

细胞处理前 PBMC 的接种控制

细胞处理之前，人类 PBMC 的接种过程通 过透射光成像监控和确认。这些细胞的直 径小于 6 µm，用透射光计数单个细胞是非 常困难的。因此，我们采用用户自定义的 分析方法，通过计算图片上覆盖面积的百 分比来计算细胞密度。这种方法计算出 19.7% 覆盖面积代表 50,000 PBMC/孔， 35.9% 覆盖面积代表 100,000 PBMC/孔， 计算比率为 1.82 ( 数据未显示 )。结果证 明，这种新的自定义数据分析方法成功识 别了细胞数量，并使每孔的面积覆盖率达 到了精确的百分比。

地塞米松处理后 LPS 或 PMA/离子霉素诱 导细胞因子释放的调节 如预期，LPS 显著诱导 IL1β

细胞处理之前，人类 PBMC 的接种过程通 过透射光成像监控和确认。这些细胞的直 径小于 6 µm，用透射光计数单个细胞是非 常困难的。因此，我们采用用户自定义的 分析方法，通过计算图片上覆盖面积的百 分比来计算细胞密度。这种方法计算出 19.7% 覆盖面积代表 50,000 PBMC/孔， 35.9% 覆盖面积代表 100,000 PBMC/孔， 计算比率为 1.82 ( 数据未显示 )。结果证 明，这种新的自定义数据分析方法成功识 别了细胞数量，并使每孔的面积覆盖率达 到了精确的百分比。

图 1 LPS 刺激下的细胞因子分泌，在 SpectraMax i3x 微孔板读板机上进行 HTRF 分析

有趣的是，在 2 µg/mL 离子毒素存在的情 况下，IL2 和 IFNγ 的分泌会显著下降 (数 据未显示) 。

无论是 LPS 还是 PMA/离子毒素处理，地塞 米松都会显著降低细胞因子的释放 ( 图 2 ) 。

图 2 地塞米松对细胞因子分泌的影响。 TNFα，IL2 和 IFNγ 的剂量效应曲线没有显示

EarlyTox 细胞完整性检测试剂盒监测 LPS， 地塞米松和 PMA/离子毒素处理对细胞活性 的影响

EarlyTox 数据是通过红色标记计算细胞数 量 ( 大小和荧光强度阈值 ) 和鉴定活/死细 胞数量来分析的。使用 SoftMax Pro 软件 中的分类功能，细胞核只标记了红色的为 活细胞，细胞核标记红色和绿色的为死细 胞。鉴定结果可以在软件中看到，细胞分 别标记成了红色 ( 活细胞 ) 和蓝色 ( 死细 胞 ) ( 图 3 和 4 ) 。

图 3 LPS + 地塞米松处理 PBMC。 利用 SoftMax Pro 软件中的分类特性，识别活细胞 (红色)，但没 有识别死细胞 (蓝色)

图 4 PMA + 离子毒素处理 PBMC。 用 SoftMax Pro 软件的分类功能，可将细胞鉴定为活细胞 ( 红 色标记 ) 或死细胞 ( 蓝色标记 ) 簇

为了进一步了解 PMA/离子毒素对细胞活性 和形态的影响，分别用 PMA 和离子毒素 加大剂量处理 PBMC。处理方法如上 ( 成 像图片未显示 ) 。如图 5 所示，加大两种 化合物浓度细胞死亡会显著增加，表明这 两种药物诱导细胞毒性。而且，数据显示 PMA 的细胞毒性几乎是离子毒素的 2 倍 ( 50 ng/mL PMA 导致 15% 死细胞，而 2 µg/mL 离子毒素导致 7% 死细胞) 。

表 1 用 EarlyTox 细胞完整性检测试剂盒检测 LPS 或 PMA/离子毒素对 PBMC 细胞活性的影响 ( 死 细胞百分比 ）

为了进一步了解 PMA/离子毒素对细胞活性 和形态的影响，分别用 PMA 和离子毒素 加大剂量处理 PBMC。处理方法如上 ( 成 像图片未显示 ) 。如图 5 所示，加大两种 化合物浓度细胞死亡会显著增加，表明这 两种药物诱导细胞毒性。而且，数据显示 PMA 的细胞毒性几乎是离子毒素的 2 倍 ( 50 ng/mL PMA 导致 15% 死细胞，而 2 µg/mL 离子毒素导致 7% 死细胞) 。

图 5 加大 PMA 和离子毒性浓度对细胞毒素的影响

Normalization of cytokine production to cell viability

整合 PBMC 活性与细胞因子分泌数据是对 生物反应进行准确分析的最后阶段。采用 EarlyTox 细胞完整性检测试剂盒检测，数 据归一化通过 HTRF 技术测定细胞因子浓 度除以活细胞数。

在图 6 中，细胞因子浓度用活细胞数表示， 药理窗口和 IC50 如表 2 所示。如预期，由 于地塞米松没有明显的细胞毒性，因此归 一化前后的药理结果是相似的。

图 6 地塞米松对归一化细胞因子分泌的影响。 。TNFα，IL2 和IFNγ 的剂量反应曲线未显示

表 2 PBMC 刺激后的药理窗口 ( 信号/背景 ) 和 IC50 值

然而，归一化 PMA/离子毒素处理的 IL2 或 IFNγ 分泌对细胞活性值的影响显示了对 PMA 浓度的依赖 ( 图 7 ) ，这点以前未检测 到。这表明细胞因子分析 ( 或其他生物标 志物 ) 与活细胞数量的归一化可以揭示相 关的生物学响应。

图 7 不同浓度 PMA/离子毒素对归一化细胞因子分析的影响。 上图 hIL2 分泌归一化为活细胞数量， 下图 hIFNγ 分泌归一化为活细胞数量

结论

本研究明确了 PMA 和离子毒素对细胞毒性 的影响，相反地塞米松没有显著的细胞毒 性。用 EarlyTox 细胞完整性检测试剂盒进 行细胞活性检测结果显示，PMA 或离子毒 素具有明显的浓度反应效应。

用 Cisbio 生物分析方法评估细胞因子分泌 和悬浮细胞如 PBMC 中药物对细胞毒性影 响的分析方法简单易用且灵敏度高。结合使 用 EarlyTox 细胞活性检测试剂盒在 SpectraMax i3x 和 MiniMax 细胞成像仪能够进行 HTRF 细胞因子的定量和细胞活性评估。

Download PDF