Application Note
Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit
- Reduce ELISA experimentation time from 260 min to 90 min with no compromise in sensitivity and reproducibility using Abcam’s SimpleStep ELISA kit
- Read an entire ELISA plate in as little as 5 seconds with the SpectraMax ABS Plus Microplate Reader
- Automatically calculate sample concentrations and plot results with SoftMax Pro Software
Introduction
Cathy Olsen | Sr. Applications Scientist | Molecular Devices
THP-1, a human monocytic cell line derived from an acute monocytic leukemia patient, is often used to study macrophage function in vitro. THP-1 cells are normally cultured in suspension, but when treated with phorbol 12-myristate 13-acetate (PMA), they differentiate into macrophages and become adherent. They also begin to release cytokines, including interleukin-8 (IL-8), which is secreted by a variety of cell types and plays a role in regulation of the acute inflammatory response.
Enzyme-linked immunosorbent assays (ELISAs) are commonly used to quantitate analytes like IL-8 in a 96-well microplate format. A typical ELISA can take over four hours to perform, but with Abcam's SimpleStep ELISA® kit, one can process an ELISA in just 90 minutes, decreasing the time spent by nearly two thirds, with no compromise in sensitivity and reproducibility (Figure 1). Here, we describe how to run the Human IL-8 SimpleStep ELISA and detect the results on the SpectraMax® ABS Plus Microplate Reader using a convenient preconfigured protocol in SoftMax® Pro Software.
Figure 1. Comparison between SimpleStep ELISA kit and conventional ELISA workflows. Abcam’s SimpleStep ELISA kit provides results in 90 minutes with its streamlined protocol. Unlike conventional ELISAs, the SimpleStep ELISA kit only requires one incubation and one wash step, reducing assay time by nearly two thirds.
Materials
- Human IL-8 SimpleStep ELISA kit (Abcam cat. #ab214030)
- THP-1 human peripheral blood monocytes (ATCC cat. #TIB-202)
- THP-1 culture medium
- RPMI-1640 Medium (ATCC cat. #30-2001)
- Benchmark Fetal Bovine Serum (Gemini cat. #100-106)
- 2-Mercaptoethanol (Sigma cat. #M6250-100mL)
- Penicillin-Streptomycin (10,000 U/mL) (ThermoFisher cat. #15140122)
- SpectraMax ABS Plus Microplate Reader (Molecular Devices cat. #ABS Plus)
Methods
THP-1 Cell Preparation
20,000 undifferentiated THP-1 suspension cells were seeded into each well of a 96-well microplate. Afterwards, THP-1 cells were treated with 2.5 ng/µL PMA, or with DMSO (negative control), diluted in culture media. Following treatment, the cells were placed in an incubator at 37°C for 48 hours. Cell supernatants were then collected and stored at -80°C until the ELISA was performed.
ELISA
On the day of the ELISA, sample supernatants were thawed, spun down at 2,000 x g for 10 minutes in a micro-centrifuge to remove debris, and diluted 1:5, 1:10, and 1:50 in the provided Sample Diluent. The Human IL-8 SimpleStep ELISA reagents and standards were prepared according to the kit’s Protocol Booklet. Samples and standards were added to the provided microplate, Antibody Cocktail was added to the wells, and the plate was sealed and incubated at room temperature for one hour on a plate shaker. Wells of the plate were then washed three times with Wash Buffer PT. TMB Substrate was added to each well, and the plate was incubated for 15 minutes in the dark on a plate shaker. Stop Solution was added to each well, and absorbance (OD) was read at 450 nm on the SpectraMax ABS Plus Microplate Reader using a preconfigured protocol in SoftMax Pro Software (see Figure 1 for workflow).
Results
A linear IL-8 standard curve with r2 = 1.000 was generated in the 'HRP and TMB' protocol in SoftMax Pro Software (Figure 2). From the standard curve, the software interpolated IL-8 concentrations of samples derived from DMSO-treated negative control cells and PMA-treated cells. Samples with insufficient dilution were flagged in the preconfigured protocol’s group table as out of range ('R') of the standard curve (Figure 3). The concentration of IL-8 from PMA-treated cells was calculated to be 3400 pg/mL, 55-fold higher than the 63 pg/mL found in DMSO-treated negative controls (Figure 4).
Figure 2. ELISA standard curve. The IL-8 standards were diluted in Sample Diluent and assayed using the Human IL-8 SimpleStep ELISA kit. A linear curve fit was applied to the standards in SoftMax Pro Software, and the generated standard curve demonstrated excellent linearity (r2 = 1.000)
Figure 3. Group table displaying calculated sample concentrations. The IL-8 concentrations of PMA-treated cells were automatically interpolated from the standard curve by the preconfigured SoftMax Pro protocol and displayed in a group table. Any samples showing an ‘R’ in the ‘R’ column had a calculated concentration that is outside the range of the standard curve, in this case indicating the need to further dilute the sample.
Figure 4. IL-8 concentrations measured in undifferentiated (DMSO control) and differentiated (2.5 ng/µL PMA) THP-1 cells. 2.5 ng/µL PMA-treated THP-1 cells produced 3400 pg/mL of IL-8 compared to the 62 pg/mL IL-8 produced by undifferentiated THP-1 cells, a 55-fold increase.
Conclusion
The SpectraMax ABS Plus Microplate Reader provides rapid and sensitive IL-8 detection with Abcam’s Human IL-8 SimpleStep ELISA kit. The SimpleStep ELISA’s proprietary antibody binding technology enables quantitation of IL-8 in THP-1 cell supernatant in as little as 90 minutes, only about a third of the time required to perform a conventional ELISA, with no compromise in sensitivity and reproducibility.
The reader’s 8-channel detector can read an entire 96-well ELISA plate in as little as 5 seconds. A preconfigured protocol in SoftMax Pro Software automatically plots a standard curve, calculates sample concentrations, and can plot results for negative controls vs. treated cells, further reducing the time needed to obtain publication-quality results.
SimpleStep ELISA® is a registered trademark of Abcam plc.
前言
利用 SpectraMax ABS Plus 微孔板读板机和 SimpleStep ELISA 试剂盒定量白细胞介素 -8
Cathy Olsen | Sr. Applications Scientist | Molecular Devices
THP-1 是人外周血的单核细胞系，最初来 源于急性单核细胞性白血病患者，常被用 来体外研究巨噬细胞功能。 它通常属于悬 浮细胞，当用 PMA 处理后可分化为贴壁 生长的巨噬细胞。THP-1 能释放许多细胞 因子，其中包括白细胞介素 -8(IL-8)，一 种能被许多类型细胞分泌的、在调节急性 炎症反应中发挥作用的细胞因子。
IL-8 与许多分析物一样可以在 96 孔板中 被 ELISA 检测定量。通常 ELISA 实验需要耗 费 4 个小时，而在 Abcam 的 SimpleStep ELISA 试剂盒帮助下实验时间可以缩短至 90 分钟，节约了近三分之二的时间，并且 无损实验的灵敏度和重复性 ( 图1 )。这里 我们介绍了如何使用 hIL-8 SimpleStep ELISA 试剂盒以及如何在 SpectraMax ABS Plus 微孔板读板机上使用 SoftMax Pro 软 件预置的便捷程序来测定 IL-8 的含量。
Figure 1. Comparison between SimpleStep ELISA kit and conventional ELISA workflows. Abcam’s SimpleStep ELISA kit provides results in 90 minutes with its streamlined protocol. Unlike conventional ELISAs, the SimpleStep ELISA kit only requires one incubation and one wash step, reducing assay time by nearly two thirds.
材料
- hIL-8 SimpleStep ELISA 试剂盒 (Abcam cat. #ab214030）
- THP-1 人外周血单核细胞 (ATCC cat. #TIB-202）
- THP-1培养基
- RPMI-1640培养基 （ATCC cat. #30-2001）
- Benchmark胎牛血清 （Gemini cat. #100-106）
- β-巯基乙醇 （Sigma cat. #M6250-100mL）
- 青-链霉素 (10,000 U/mL) （ThermoFisher cat. #15140122)
- SpectraMax ABS Plus微孔板读板机 （Molecular Devices cat. #ABS Plus）
方法
准备 THP-1 细胞
96 孔板每孔中预先加入 2 万个悬浮的未 分化的 THP-1 细胞，随后用2.5 ng/μL 的 PMA 或 DMSO（阴性对照）37℃ 处理细 胞 48 小时。处理时间结束后收集细胞上 清 -80℃ 冻存直至 ELISA 检测。
ELISA
ELISA 实验前需融化样品，2000 g 离心 10 分钟去除细胞碎片，上清液用试剂盒提 供的稀释液按照 1:5，1:10 以及 1:50 比例 稀释。按照试剂盒说明书配制好标准品 后，将样品与标准品加入试剂盒提供的微 孔板中。然后每孔加入抗体混合物，孔板 密封好后放入摇床室温震荡 1 小时。1 小 时后用 Wash Buffer PT 洗涤每孔 3 遍， 然后加入底物 TMB，暗室内置于摇床上震 荡 15 分钟显色。15 分钟后加入终止液终 止反应，并于 SpectraMax ABS Plus 微 孔板读板机上测定 450nm 处吸光度。这 一过程可在 SoftMax Pro 软件预置的便捷 程序中实现 ( 参见图 1 工作流程 )。
结果
利用 SoftMax Pro 软件预置的“HRP and TMB”模板可以得到 IL-8 的线性标准曲 线，R2 达到 1000 ( 图 2 )。随后软件根据 标准曲线可以获得 PMA 处理和阴性对照 样品中 IL-8 的浓度。因稀释度不够而超 出标准曲线线性范围的样品在结果列表中 被标记为“R”( 图 3 )。图 4 结果显示了 经软件计算后 PMA 处理后的细胞 IL-8 的 含量为 3400 pg/mL，比用 DMSO 处理的 阴性对照 (63 pg/mL) 高 55 倍。
Figure 2. ELISA standard curve. The IL-8 standards were diluted in Sample Diluent and assayed using the Human IL-8 SimpleStep ELISA kit. A linear curve fit was applied to the standards in SoftMax Pro Software, and the generated standard curve demonstrated excellent linearity (r2 = 1.000)
Figure 3. Group table displaying calculated sample concentrations. The IL-8 concentrations of PMA-treated cells were automatically interpolated from the standard curve by the preconfigured SoftMax Pro protocol and displayed in a group table. Any samples showing an ‘R’ in the ‘R’ column had a calculated concentration that is outside the range of the standard curve, in this case indicating the need to further dilute the sample.
Figure 4. IL-8 concentrations measured in undifferentiated (DMSO control) and differentiated (2.5 ng/µL PMA) THP-1 cells. 2.5 ng/µL PMA-treated THP-1 cells produced 3400 pg/mL of IL-8 compared to the 62 pg/mL IL-8 produced by undifferentiated THP-1 cells, a 55-fold increase.
结论
SpectraMax ABS Plus 微孔板读板机和 Abcam hIL-8 SimpleStep ELISA 试剂盒 提供了一种快速、灵敏的检测 IL-8 的方 法。Abcam SimpleStep ELISA 试剂盒独 有的抗体结合技术，使得从 THP-1 细胞上 清中测定 Il-8 含量的时间可以缩短至 90 分钟，比传统 ELISA 方法节约了近三分之 二的时间，并且实验结果保持了原有的灵 敏度和重复性。
SpectraMax ABS Plus 微孔板读板机所采 用的8通道检测器能够极大地缩短用户获 得实验数据的时间，其整块 96 孔 ELISA 板的读板时间仅为 5 秒。此外 SoftMax Pro 软件中预置的模板，还可以根据实验结果 自动计算样品浓度和绘制标准曲线，并且 能够将阴性对照曲线与实验结果曲线绘制 在同一张图谱上加以比较，进一步减少用 户分析实验数据的时间，帮助用户获得高 质量、能发表的实验结果。