Test de mort cellulaire : libération du chromium pour mesurer la cytotoxicité
English
Share
Overview
Source : Frances V. Sjaastad1,2, Whitney Swanson2,3, et Thomas S. Griffith1,2,3,4
1 Microbiologie, immunologie et programme d’études supérieures en biologie du cancer, Université du Minnesota, Minneapolis, MN 55455
2 Center for Immunology, Université du Minnesota, Minneapolis, MN 55455
3 Département d’urologie, Université du Minnesota, Minneapolis, MN 55455
4 Centre du cancer maçonnique, Université du Minnesota, Minneapolis, MN 55455
Une des principales fonctions des cellules du système immunitaire est d’enlever les cellules cibles qui ont été infectées par des virus ou ont subi une transformation en une cellule tumorale. Les tests in vitro pour mesurer la capacité cytotoxique des cellules immunitaires sont un aliment de base dans les laboratoires depuis de nombreuses années. Ces essais ont été utilisés pour déterminer la capacité des lymphocytes T, des cellules NK, ou de toute autre cellule immunitaire à tuer les cellules cibles d’une manière spécifique à l’antigène ou non spécifique. Les ligands de la mort (p. ex. Fas ligand ou TRAIL), les cytokines (p. ex. IFNg ou TNF) ou les granules cytotoxiques (p. ex., perforine/granzyme B) exprimés par les cellules effectrices sont quelques façons d’intenter l’induit de la mort cellulaire cible. Avec l’explosion de la recherche d’immunothérapie de tumeur ces dernières années, il y a l’intérêt croissant dans trouver des agents pour augmenter l’activité cytotoxique des cellules immunitaires pour améliorer des résultats patients. Inversement, certaines maladies sont marquées par l’activité surexubérante de l’activité cytotoxique des cellules immunitaires, ce qui entraîne des efforts pour identifier les agents pour tempérer ces réponses. Ainsi, avoir un test dans lequel l’utilisateur peut facilement intégrer un certain nombre de cellules effectrices différentes, cellules cibles, et / ou modificateurs de réponse dans la conception expérimentale peut servir de moyen précieux d’évaluer rapidement la capacité cytotoxique des cellules effectrices et / ou la réactivité de la cellule cible.
Ces essais in vitro impliquent le mélange de différentes populations cellulaires, ainsi que l’utilisation d’un nombre relativement faible de cellules effectrices et cibles. Ainsi, l’une des nécessités de l’analyse est d’étiqueter les cellules cibles d’une manière qui peut facilement être détectée et quantit, permettant à l’utilisateur de déterminer ensuite la « lyse spécifique pour cent » médiée par les cellules effectrices. La radioactivité – en particulier, le chrome 51 (51Cr) sous la forme de Na251CrO4– est un moyen peu coûteux d’étiqueter rapidement et non spécifiquement les protéines cellulaires dans les cellules cibles (1). L’étiquetage court et les temps d’assiduité total réduisent le risque de changements significatifs dans le nombre et/ou le phénotype des cellules cibles, ce qui pourrait influencer le résultat de l’effort. Lors de la perte de l’intégrité de la membrane des cellules cibles en raison de l’activité cytotoxique des cellules effectrices, les 51protéines cellulaires étiquetées Cr dans les cellules cibles sont libérées dans le supernatant de culture, devenant disponibles pour Quantification. Comme avec tout analyse examinant la fonction des cellules immunitaires in vitro,il ya un certain nombre de considérations importantes à envisager d’améliorer la performance de l’expérience. Une des caractéristiques les plus critiques est d’utiliser l’effecteur sain (pour l’activité cytotoxique maximale) et la cible (pour la réactivité maximale et la mort spontanée minimale /51Cr libération) cellules. Le contact entre l’effet et les cellules cibles est nécessaire (ce qui conduit à l’utilisation courante de plaques rondes de 96 puits pour encourager le contact cellule-cellule) (2). Enfin, l’analyse des données dépend de l’inclusion de populations de cellules cibles de contrôle positives et négatives.
Le protocole suivant exposera les étapes pour effectuer un jeu standard de libération de 51Cr pour mesurer la capacité cytotoxique d’une population de cellules effectrices, bien qu’une version non radioactive utilisant europium ait été récemment développée. 51 Annonces Cr est un puissant émetteur de rayonnement. Par conséquent, l’utilisation de cet analyse nécessite une formation appropriée en matière de sécurité des rayonnements, un espace de laboratoire dédié, un compteur gamma et l’élimination d’échantillons radioactifs.
La séquence générale des événements dans cet état d’œil est la suivante : 1) préparer 51cibles étiquetées Cr; 2) préparer les cellules effectrices et les ajouter à la plaque pendant que les cellules cibles sont étiquetées; 3) ajouter des cibles étiquetées à la plaque; 4) plaque incubée; 5) les supernatants de récolte ; et 6) analyser les données après l’exécution d’échantillons sur le comptoir. Les échantillons sont généralement préparés en triple, puis en moyenne pour tenir compte de toute différence subtile de pipetage.
Un EPI approprié est important pour cet avertissement. Plus précisément, l’utilisateur doit porter une blouse de laboratoire et des gants. Des lunettes de sécurité peuvent être requises en fonction du laboratoire ou de l’établissement. Il devrait y avoir un large blindage au plomb pour un stockage sûr et l’utilisation de la 51Cr pendant toutes les étapes. Enfin, il devrait y avoir un espace de laboratoire et de l’équipement dédiés réservés à l’utilisation de 51Cr, y compris toutes les signalisations appropriées pour indiquer où les échantillons avec 51Cr sont conservés et un compteur Geiger équipé d’une sonde gamma pour étudier l’espace pour possible contamination.
Dans cet exercice de laboratoire, nous déterminerons la capacité des cellules mononucléaires périphériques humaines de sang (PBMCs), (CpG stimulées contre non stimulées) pour tuer des cellules de mélanome, utilisant la ligne humaine de cellules de mélanome WM793 comme modèle et l’essai de libération de chrome.
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.