세포 사멸 분석: 세포 독성 능력의 크롬 방출 분석
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출처: 프랜시스 V. 자아스타드1,2,휘트니 스완슨2,3,토마스 에스 그리피스1,2,3,4
1 미생물학, 면역학 및 암 생물학 대학원 프로그램, 미네소타 대학교, 미니애폴리스, MN 55455
2 면역학 센터, 미네소타 대학, 미니애폴리스, MN 55455
3 미네소타 대학교 비뇨기과, 미니애폴리스, MN 55455
4 Masonic 암 센터, 미네소타 대학, 미니애폴리스, MN 55455
면역 계통의 세포의 주요 기능 의 한은 바이러스에 감염되었거나 종양 세포로 변환을 겪은 표적 세포를 제거하는 것입니다. 면역 세포의 세포 독성 용량을 측정하기위한 체외 분석을 수년 동안 실험실에서 주식으로 되어 왔습니다. 이러한 소약은 T 세포, NK 세포 또는 항원 특이적 또는 비특이적 방식으로 표적 세포를 죽이는 다른 면역 세포의 능력을 결정하는 데 사용되었습니다. 데스 리간드(예를 들어, 파스 리간드 또는 트레일), 사이토카인(예를 들어, IFNg 또는 TNF), 또는 이펙터 세포에 의해 발현된 세포독성 과립(즉, 페포린/그란자임 B)은 표적 세포 사멸을 유도할 수 있는 몇 가지 방법이다. 최근 몇 년 동안 종양 면역 요법 연구의 폭발로 환자 결과를 개선하기 위해 면역 세포의 세포 독성 활성을 증가시키는 에이전트를 찾는 데 관심이 증가하고 있습니다. 반대로, 몇몇 질병은 면역 세포 중독 활동의 과잉 활동으로 특징지어집니다, 이 반응을 강화하는 에이전트를 확인하는 노력의 결과로. 따라서, 사용자가 실험 설계에 임의의 상이한 이펙터 세포, 표적 세포 및/또는 반응 수정자를 쉽게 통합할 수 있는 분석체는 표적 세포의 세포 독성 용량 및/또는 반응성을 신속하게 평가하는 귀중한 수단으로 작용할 수 있다.
이러한 시험관 내 아세약은 다른 세포 집단의 혼합뿐만 아니라 이펙터 및 표적 세포 모두의 상대적으로 낮은 수를 사용하는 것을 포함한다. 따라서, 분석의 한 가지 필요성은 표적 세포를 쉽게 검출하고 수량화할 수 있는 방식으로 라벨을 부착하여 사용자가 이펙터 세포에 의해 매개되는 ‘백분율 특이용 용해’를 결정할 수 있도록 하는 것이다. 방사능 -특히, 크롬 51(51Cr)의 형태로 Na251CrO4– 표적 세포 내의 신속하고 비구체적으로 라벨 세포 단백질을 저렴한 방법입니다 (1). 짧은 라벨링 및 총 분석 시간은 분석의 결과에 영향을 미칠 수 있는 표적 세포의 수 및/또는 표현형에 있는 중요한 변경에 대한 잠재력을 감소시킵니다. 이펙터 세포의 세포 독성 활성의 결과로 표적 세포의 막 무결성의 상실시, 대상 세포 내의 51Cr 라벨 세포 단백질은 배양 상수로 방출되어 양수에 사용할 수 있게 된다. 시험관 내면역 세포의 기능을 검사하는 분석과 마찬가지로 실험의 성능을 개선하는 것을 고려하는 중요한 고려 사항이 많이 있습니다. 가장 중요한 특징 중 하나는 건강한 이펙터 (최대 세포 독성 활성)와 대상 (최대 반응성과 최소한의 자발적 사망 /51Cr 방출) 세포를 사용하는 것입니다. 이펙터 및 표적 세포 접촉이 요구된다(세포-세포 접촉을 장려하기 위해 라운드 하단 96-웰 플레이트의 일반적인 사용으로 이어지도록 유도) (2). 마지막으로, 데이터 분석은 양수 및 음극대상 세포 집단의 포함에 의존한다.
다음 프로토콜은 유로피움을 이용한 비방사성 버전이 최근에 개발되었지만, 이펙터 세포의 집단의 세포 독성 능력을 측정하기 위한 표준 51Cr 방출 분석기를 수행하기 위한 단계를 설명할 것입니다. 51 Cr은 강력한 γ 방사선 방출기입니다. 따라서 이 분석법의 사용에는 적절한 방사선 안전 교육, 전용 실험실 공간, 감마 카운터 및 방사성 시료 의 폐기가 필요합니다.
이 분석기의 일반적인 이벤트 순서는 다음과 같습니다: 1) 51Cr 라벨 대상을 준비; 2) 대상 세포가 라벨링하는 동안 이펙터 세포를 준비하고 접시에 추가; 3) 플레이트에 레이블이 붙은 대상을 추가합니다. 4) 인큐베이션 플레이트; 5) 수퍼나일체 수확; 및 6) 카운터에서 샘플을 실행한 후 데이터를 분석합니다. 샘플은 일반적으로 triplicate에서 제조된 다음 미묘한 파이펫팅 차이를 설명하기 위해 평균화됩니다.
적절한 PPE는이 분석에 중요합니다. 특히, 사용자는 실험실 코트와 장갑을 착용해야합니다. 안전 안경은 실험실 이나 기관에 따라 필요할 수 있습니다. 모든 단계에서 51Cr을 안전하게 보관하고 사용할 수 있는 충분한 리드 차폐가 있어야 합니다. 마지막으로, 51Cr을 사용하는 전용 실험실 공간과 장비가 있어야 하며, 51Cr을가진 시료가 보관되는 위치를 나타내는 모든 적절한 사이니지와 감마 프로브가 장착된 가이거 카운터를 포함하여 오염 가능한 오염을 위한 공간을 조사해야 합니다.
본 실험실 운동에서는 인간 흑색종 세포를 죽이는 인간 말초 혈액 단핵세포(PBMC), (CpG 자극 대 자극) 인간 흑색종 세포주 WM793를 모델및 크롬 방출 분석으로 결정할 것이다.
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.