细胞死亡测定：细胞毒性能力的铬释放测定
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Overview
资料来源：弗朗西斯·萨亚斯塔德1，2，惠特尼·斯旺森2，3和托马斯·格里菲斯1，2，3，4
1明尼苏达大学明尼阿波利斯分校微生物学、免疫学和癌症生物学研究生课程，MN 55455
2明尼苏达大学明尼阿波利斯分校免疫学中心，MN 55455
3明尼苏达大学泌尿科，明尼阿波利斯，MN 55455
4共济会癌症中心，明尼苏达大学明尼阿波利斯分校，MN 55455
免疫系统细胞的主要功能之一是去除已经感染病毒或已经转化为肿瘤细胞的目标细胞。多年来，用于测量免疫细胞细胞毒性能力的体外检测一直是实验室的主食。这些测定用于确定T细胞、NK细胞或任何其他免疫细胞以抗原特异性或非特异性方式杀死目标细胞的能力。由效应细胞表达的死亡配体（例如，Fas配体或TRAIL）、细胞因子（如IFNg或TNF）或细胞毒性颗粒（即穿孔素/颗粒B）是诱导靶细胞死亡的一些方法。随着近年来肿瘤免疫治疗研究的爆炸式增长，人们越来越有兴趣寻找增加免疫细胞细胞细胞毒性活性的制剂，以改善患者的疗效。相反，一些疾病的特点是免疫细胞细胞毒性活性过度，导致努力识别抑制这些反应的病原体。因此，通过测定，用户可以轻松地将任意数量的不同效应细胞、靶细胞和/或响应修饰剂集成到实验设计中，可以作为快速评估效应细胞细胞的细胞毒性和/或目标单元格的响应能力。
这些体外检测涉及混合不同的细胞群，以及使用相对较少的效应细胞和目标细胞。因此，检测的一个必要条件是以易于检测和定量的方式标记目标细胞，允许用户确定由效应细胞介导的”百分比特异性莱沙”。放射性 – 特别是，铬51（51Cr）的形式Na251CrO4– 是一种廉价的方式，快速和非具体标记细胞蛋白在目标细胞 （1）。短标记和总测定时间降低了目标细胞的数量和/或表型发生重大变化的可能性，这可能会影响测定结果。当由于效应细胞的细胞毒性活性而使目标细胞的膜完整性丧失时，目标细胞内的51个Cr标记细胞蛋白被释放到培养上清液中，可用于定量。与任何在体外检查免疫细胞功能的测定一样，需要考虑提高实验性能。其中一个最关键的特征是使用健康效应器（用于最大的细胞毒性活性）和目标（用于最大响应性和最小自发死亡/51Cr释放）细胞。需要效应器和目标细胞接触（导致普遍使用圆底96孔板来鼓励细胞-细胞接触） （2）。最后，数据分析取决于纳入正负对照细胞群。
以下协议将概述执行标准51Cr 释放测定以测量效应细胞群的细胞毒性能力的步骤，尽管最近开发了使用 Europium 的非放射性版本。51Cr 是一个强大的 -辐射发射器。因此，使用这种检测需要适当的辐射安全培训、专用的实验室空间、伽马计数器和放射性样品的处置。
此测定中的事件总序列为：1） 准备51个 Cr 标记的目标;2） 在目标细胞标记时制备效应细胞并添加到板中;3） 将标记的目标添加到板中;4）孵育板;5） 收获超生物;6） 在计数器上运行样本后分析数据。样品通常以三联，然后平均，以解释任何细微的移液差异。
适当的 PPE 对于此测定非常重要。具体来说，用户应穿着实验室外套和手套。根据实验室或机构，可能需要安全眼镜。所有步骤都应有足够的铅屏蔽，以便安全存储和使用51Cr。最后，应留出专用实验室空间和设备，以便使用51Cr，包括所有适当的标牌，以指示存放51Cr 的样品的位置，以及配备伽马探测器的盖革计数器，以便尽可能测量空间污染。
在本实验练习中，我们将确定人类外周血单核细胞（PBMC）、（CpG刺激与未刺激）杀死黑色素瘤细胞的能力，使用人类黑色素瘤细胞系WM793作为模型和铬释放测定。
Procedure
Results
In this example, effector cells stimulated with CpG (Figure 1, black circles) killed the target cells more effectively, as the ratio of effector cells to target cells increased. This increase was not observed in the unstimulated PBMCs (white circles), indicating that CpG stimulation is necessary for the observed increase in target cell lysis.
Figure 1: 51Cr assay scatter plot: Tumoricidal activity by human PBMCs, unstimulated (white circles) and after stimulation with CpG (black circles), tested at different effector: target cell ratios (E: T) ratios (ranging from. 50:1 to 1.5:1).
Applications and Summary
The assay described here has considerable flexibility, as a variety of effector and target cells can be used depending on the question being asked. For example, effector cell specificity can be determined by using different target cells or the mechanism of effector cell killing can be determined by using cells deficient in specific proteins or using protein specific inhibitors. A major problem with the 51Cr release assay is the potential for a high spontaneous release rates by the target cells. When cultured alone (without effector cells), the spontaneous release of 51Cr by the target cells should ideally be no more than 30% of the total ("maximal") release by the target cells immediately lysis. Higher spontaneous release rates may be due to using unhealthy target cells, either due to poor health (e.g., extended culture of a cell line) or an overly long labelling period.
References
- Brunner, K. T., Mauel, J., Cerottini, J. C. and Chapuis. B. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology, 14 (2):181-196, (1968).
- Kemp, T. J., B. D. Elzey, and T. S. Griffith. Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/Apo-2L-mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation. The Journal of Immunology, 171 (1): 212-218, (2003).
Transcript
In this video you will observe how to perform the chromium release assay and determine the cytotoxic potential of the effector cells.
Immune cells are responsible for identifying and removing potentially harmful cells, like cancer or virus-infected cells, from the body, which is an integral part of the immune response. Several immune cells, like T-cells and NK cells, possess a property known as cytotoxic potential, which is the ability to identify target cells and secrete proteins that induce protein degradation, lysis, and death of those target cells. Quantifying cytotoxic potential is critical for measuring immune cell activation and potency, and the chromium release assay is commonly used for this purpose.
This method enables users to compare cytoxicity induced by specific types of immune cells under different conditions, which is valuable for studying cancer immunotherapy and immunity related diseases. To begin, the target cells, like cancer cells, are incubated with a radioactive isotope, chromium 51, which is taken up by the cells. Next, these radio labeled cells are co-cultured with the isolated immune cells of interest, also called the effector cells, in a round bottom, 96- well plate to facilitate interaction between the two cell types.
The overall setup of the assay involves incubating a specific number of target cells with different concentrations of the immune cells, along with appropriate controls. The co-culture allows the effector cells to induce apoptosis and lysis in the target cells, resulting in the release of the intracellular chromium 51 into the supernatant. Then, at a preoptimized time point, the supernatant containing the released chromium is harvested from all the wells. The chromium 51, being radioactive, spontaneously undergoes radioactive decay to emit gamma radiation. The gamma radiation levels in the supernatants from all the wells in the assay plate represent a quantifiable output of the lysis of the target cells. This is measured using a gamma counter, which is then used to determine the cytotoxic potential of the immune cells.
To begin, the target cells, human melanoma cell line WM793 in this example, are prepared into a single cell suspension. To do this, first remove the media from the tissue culture flask and wash the cells with five milliliters of 1X PBS. Decant the PBS and then add one milliliter of trypsin to the plate for approximately two minutes. Gently tap the flask to loosen the cells from the flask surface and then add five milliliters of RPMI media to the flask. Pipette the media up and down to collect the cells and add this suspension to a 15 milliliter conical tube.
Place the tube in the centrifuge for five minutes at 1200 RPM. Next, remove the media from the tube making sure not to disrupt the cell pellet. Gently flick the bottom of the tube to disrupt the cell pellet and add 10 milliliters of media to the tube. Then, gently pipette the media up and down to bring the cells into suspension. Next, determine the cell concentration using a hemocytometer and transfer two milliliters of the original cell suspension to a new 15 milliliter conical tube. Place the tube into a centrifuge and pellet the cells at 12 hundred RPM for five minutes. After centrifugation, pour the excess media out of the tube into a waste container. Briefly vortex the tube to resuspend the cell pellet in the small volume of medium left behind.
Next, prepare to use Chromium 51 by moving to a lab space dedicated for this particular radioactivity. There should be ample lead shielding for safe storage and use of the Chromium 51 during all steps, as well as proper signage to indicate where samples with Chromium 51 are being kept. A Geiger counter equipped with a pancake probe is also necessary to serve in the space for possible contamination.
Once set up for the proper use of radioactivity, add 100 microcuries of Chromium 51 directly to the target cell suspension. Then, add a small piece of radioactive tape to the tube to indicate that the sample and tube are now radioactive. Place the tube in a 37 degree celsius incubator with a lead shield and incubate for an hour, flicking the tube every 15 to 20 minutes.
While the target cells are labeling, prepare a single cell suspension of effector cells. In this example, human peripheral blood mono nuclear cells, or PDMCs, were isolated from whole blood by standard density gradient centrifugation to a concentration of 5 times 10 to the 6th. Transfer this effector cell suspension into a disposable reagent reservoir and then add 200 microliters of this suspension into each well of row B in a 96-well round-bottom plate. Next, add 100 microliters of RPMI to each well in row C through G of the plate.
Now, begin performing serial dilutions of the PBMCs to have a range of effector cell numbers by first removing 100 microliters of the cells in the wells in row B and adding this to row C. Then, further dilute the effector cells by transferring 100 microliters of cells from row C to row D. Continue the serial dilution. Once row G is reached, move 100 microliters from the wells to leave a final volume of 100 microliters in each well in that row. Next, add 100 microliters of tissue culture medium to the wells in row A to serve as a control for the spontaneous release of Chromium 51 from the target cells, as no effector cells should be added to this row. Then, place a plate into a 37 degree celsius incubator until the target cells are ready to be added.
After the incubation period, remove the target cells from the incubator and wash with 5 milliliters of FBS to remove any excess Chromium 51. Then, place the tube in a designated centrifuge and spin at 1200 rpm for 5 minutes. Remove the radioactive FBS wash into an appropriate waste container and repeat the wash step by resuspending the pellet in a fresh 5 milliliters of FBS. Place the tube in a designated centrifuge and spin the cells again at 1200 rpm for 5 minutes. Remove the second wash and check the pellet for incorporated radioactivity using a Geiger counter. Finally, Resuspend the pellet in 10 milliliters of complete medium and pour the Chromium 51 labeled, target cell suspension into a disposable reagent reservoir. Then, add 100 microliters of these labeled target cells to every well of the 96-well effector cell plate. Next, add 100 microliters of 1% NP-40 in water to the wells in row H to lyse all the target cells this each row. These wells will be used as a control to determine the total counts per minute, or cpm.
Now that the plate is prepared, secure the lid by adding a small piece of tape to the each side of the plate and place a piece of radioactive tape on the lid to indicate it contains chromium 51. Then, place the plate in a centrifuge marked to handle radioactive samples. If only one experimental plate is being used, add a balance plate to the centrifuge. Set the centrifuge to 1200 rpm, and bring the plate up to speed. Once at the speed, stop the machine. Remove the plate from the centrifuge. Then, place the plate in a 37 degree celsius incubator with a small piece of lead shielding over the plate for additional safety. Incubate for 16 hours to allow the target cells to lyse.
At the end of the incubation period, carefully remove the tape around the edge of the plate, and remove the lid. Next, place the harvesting frame on the plate making sure to confirm the small filter discs are in place for each of the cotton plugs. Now, slowly and gently press the cotton plugs into the wells. After approximately ten seconds, release the pressure on the cotton plugs, and then transfer the cotton plugs to tube strips. Place each of these tubes into a secondary FACS tube. Finally, load the FACS tubes onto a gamma counter and run the samples to quantitate the amount of chromium 51 released in each condition. Carefully record the order in which the tubes were loaded into the counter.
Here, unstimulated PBMCs were added to the first 3 lanes and CPG stimulated PMBCs were added to lanes 4 through 6. In this example, the counts per minute were entered into the cells of a spreadsheet in the same manner as the samples were laid out in the original plate and the averages of the triplicates were calculated. For example, for the first condition, cells A1, A2, and A3 were averaged in cell I3. Once the averages are determined, the percent of specific lysis for each condition can be calculated using this formula. For example, to calculate the percent specific lysis for the unstimulated cells that had a ratio of 50 to 1 effector cells to target cells the spontaneous CPM, which in this example, is 1164.67, was subtracted from the experimental CPM, 1129. 67. This number can then be divided by the difference between the maximum CPM and the spontaneous CPM, and then multiplied by 100 to give the percent specific lysis. This is then calculated for each condition. These data can then be graphed to show comparison of the E to T ratio with the percent specific lysis for both the unstimulated PBMCs, and the CPG stimulated PBMCs. In this example, effector cells stimulated with CPG more effectively killed target cells as the ratio of effector cells to target cells increased. This increase was not observed in the unstimulated PBMCs, indicating that CPG stimulation is necessary for the observed increase in target cell lysis.