Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

Procedure Overview

The typical ⁵¹Cr-release assay for measuring cell death involves the following steps:

1. First, the target cells are labeled with Na₂[⁵¹Cr]O₄. This distinguishes them from the effector cells in the assay.
2. While the target cells are labeling, the effector cells are collected and, using the serial dilution technique, a decreasing titration of the effector cells is generated in a round bottom 96-well assay plate.
3. At the end of the target cell labelling, the cells are first washed and then a fixed number of cells are added to the assay plate that already contains a series of effector cells dilutions.
4. Next, the target-effector cell mix is incubated for a defined period of time to allow for sufficient cell interaction with the target cells, to mediate cell lysis.
5. Finally, the culture supernatants are harvested and collected into tubes. The ⁵¹Cr amount is quantitated using a gamma counter.
6. At the end, the data is collected and used to calculate the "percentage specific cell lysis" of the target cells.

1. Labeling Target Cells with ⁵¹Cr

1. To begin, prepare the target cells, (here- human melanoma cell line WM793), into a single cell suspension.
2. To do this, first remove the media from the tissue culture flask.
3. Then, wash the cells with 5 mL of 1X PBS.
4. Trypsinize the cells by adding 1 mL of trypsin to the plate for ~2 min.
5. Gently tap the flask to loosen the cells from the flask surface.
6. Add 5 mL of RPMI media to the flask and pipet the media up and down to detach the cells.
7. Collect the cell suspension into a 15 mL conical tube and centrifuge the cell suspension for 5 min at 1200 rpm. Decant the supernatant.
8. Add 10 mL of media to the pellet and gently pipet the media up and down to bring the cells into suspension.
9. Determine the cell concentration using a hemocytometer.
10. Transfer 1x10⁶ cells into a new 15 mL conical tube.
11. Centrifuge the cell suspension for 5 min at 1200 rpm and decant the supernatant.
12. Briefly vortex the tube to resuspend the cell pellet in the small volume of medium left behind.
13. Add 100 µCi of ⁵¹Cr directly to the WM793 target cell suspension.
    Note: There should be a dedicated lab space set up for the particular radioactivity. In addition, there should be ample lead shielding for safe storage and use of the ⁵¹Cr during all steps as well as proper signage to indicate where samples with ⁵¹Cr are being kept. A Geiger counter equipped with a pancake probe is also necessary, for surveying the space for possible contamination.
14. Add a small piece of radioactive tape to the tube to indicate that the tube is now radioactive.
15. Place the tube in a 37°C incubator with a lead shield and incubate for 1 h. Flick the tube every 15-20 minutes to increase target cell uptake of the chromium.
16. After the incubation period, wash the target cells with 5 mL of FBS to remove any excess ⁵¹Cr.
17. Centrifuge the cells at 1200 rpm for 5 min. Decant radioactive FBS wash into appropriate waste container.
18. Resuspend the pellet and wash a second time with FBS. Check the pellet for incorporated radioactivity using a Geiger counter.
19. Resuspend the pellet in 10 mL complete medium, achieving a cell concentration of 10⁵ cell/mL.
    Note:The cell counting step after the ⁵¹Cr labelling is omitted here largely for the safety reasons. It can be assumed that the WM793 cell concentration is same as it was prior to the ⁵¹Cr labelling.

2. Preparing Effector Cells

1. A variety of effector cells can be used, such as, human or mouse T cells and NK cells. In this example, PBMCs, isolated from whole blood by standard density gradient centrifugation (to a concentration of 5x10⁶), were used.
2. To begin, add 100 µL of tissue culture medium to every well of one of the rows (here row A, See Table 1) of a 96-well round-bottom plate. Here, no effector cells are added, and they will serve as the 'blank' for determining the "minimum/spontaneous ⁵¹Cr release" from the target cells.

Table 1: ⁵¹Cr release assay layout: Row A- Target cells (10⁴ cells/ 100 µL) incubated with media only (generates 'blank' for determining the "minimum/spontaneous ⁵¹Cr release"). Rows B to C- wells containing different effector: target cell (E:T) ratios, ranging from 50:1 to 1.5:1. Row H- target cells (10⁴ cells/100 µL) incubated with 1% NP-40 (NP-40 lyses the target cells, generating "maximum or total ⁵¹Cr release"). Every E:T ratio is tested in triplicate for every experimental condition. Column 1-3- unstimulated PBMCs and column 4-6- CpG-stimulated PBMCs.

3. Next, generate a 2X serial cell dilution of the PBMCs (in triplicate for each experimental condition), to obtain an effector cell concentration ranging from 5x10⁵ to 15,625 cells/100 µL of media (here rows B to G).
   Note: In this example, the starting effector: target cell (E: T) ratio is be 50:1. However, this ratio can be adjusted depending on the experimental specifics.
4. Leave the last row empty (here row H) by not adding any effector cells to these wells. (This row will be used to generate the "total counts/min, or (c. p. m)" or the "maximum ⁵¹Cr release").
5. Place the plate into 37°C incubator until the target cells are ready to be added.

3. Adding ⁵¹Cr Labeled WM793 Target Cells to the Assay

1. After the incubation period, remove the target cells from the incubator and wash with 5 mL of FBS to remove any excess ⁵¹Cr.
2. Centrifuge the cells at 1200 rpm for 5 min, using a designated centrifuge.
3. Remove the FBS wash (radioactive supernatant) into an appropriate waste container.
4. Repeat the wash step by resuspending the pellet in a fresh 5 mL of FBS.
5. Centrifuge the cells again at 1200 rpm for 5 min.
6. Finally, resuspend the pellet in 10 mL of complete medium.
7. Pour the ⁵¹Cr-labelled WM793 cell suspension (10⁵ cells/mL) into a disposable reagent reservoir.
8. Then, add 100 μL of these labeled target cells, to each well of the 96-well effector cell plate, using a multi-channel pipette.
9. Next, add 100 μL of 1% NP-40 (in water) to the row of wells that are devoid of any effector cells (here row H). 1% NP-40 will lyse the target cells, and thus these wells will serve as controls to determine the "total counts/min, or (c. p. m)" or the maximum ⁵¹Cr release.
10. Secure lid to plate by adding a small piece of gas-permeable tape to each side of the plate and place a piece of radioactive tape on the lid to indicate it contains ⁵¹Cr.
11. Briefly, centrifuge the plate at 1200 rpm. If only one experimental plate is being used, add a balance plate to the centrifuge.
    Note: It is important to use a centrifuge marked to handle radioactive samples.
12. Remove the plate from the centrifuge.
13. Place the plate in a 37°C incubator with a small piece of lead shielding over the plate for additional safety. Incubate for 16 h to allow for target cell lysis.
    Note: The incubation period can vary from 4 to 18 h depending on the effector cells used and potential mechanism of killing employed.

4. Harvesting the Supernatants

1. At the end of the incubation period, carefully remove the tape around the edge of the plate and remove the lid.
2. Next, place the harvesting frame on the plate, making sure to confirm that the small filter discs are in place for each of the cotton plugs. This will ensure the collection of a cell-free supernatant.
3. Now, slowly and gently press the cotton plugs into the wells.
4. After approximately 10 seconds, release the pressure on the cotton plugs and then transfer the cotton plugs to tube strips.
5. Place each of these tubes into a secondary FACS tube.
6. Finally, load FACS tubes onto gamma counter and run the samples to quantitate the amount of ⁵¹Cr released in each condition. Samples are typically measured for 1 minute, allowing for an easy determination of the "counts/minute".
7. Carefully, record the order in which the tubes were loaded into the counter.

5. Data Analysis

1. Here, unstimulated PBMCs were added to the first three columns, and CpG-stimulated PBMCs (CpG ODN, (1 µg/mL) for 24 h) were added to columns 4-6. See Table 2.

	1	2	3	4	5	6	7	8
A	1251	1157	1086	917	1118	1140
B	1832	1986	1971	7629	7913	8180
C	1677	1739	1428	5454	5055	5268
D	1638	1552	1734	4239	3582	3786
E	1658	1580	1339	2818	2623	2750
F	1579	1472	1483	2028	1779	1769
G	1326	1325	1184	1801	1654	1565
H	9220	9367	8067	8774	9647	8236
I	Average of A1,A2,A3 ->		1164.67	Spontaneous c.p.m		1058.33
J	Average of B1,B2,B3 ->		1929.67	9.91%		7907.33	87.50%	50:1
K	Average of C1,C2,C3 ->		1614.67	5.83%		5259	53.67%	25:1
L	Average of D1,D2,D3 ->		1641.33	6.17%		3869	35.91%	12.5:1
M	Average of E1,E2,E3 ->		1525.67	4.68%		2730.33	21.36%	6.25:1
N	Average of F1,F2,F3 ->		1511.33	4.49%		1858.67	10.22%	3.12:1
O	Average of G1,G2,G3 ->		1278.33	1.47%		1673.33	7.86%	1.56:1
P	Average of H1,H2,H3 ->		8884.67	Maximum c.p.m		8885.67
				Unstim PBMC			CpG-stim PBMC	E:T ratio

Table 2: ⁵¹Cr release assay data: 'Counts per minute' / 'c. p. m' data values, average c. p. m values, and calculated percent specific lysis values.

2. The collected data (counts per minute, i.e. c.p.m) were entered into the cells of a spreadsheet in the same manner as the samples were laid out in the original plate.
3. First, the averages of the triplicates were calculated. Table 2 - cells I3 to P3, for unstimulated PBMCs and cells I6 to P6, for CpG-stimulated PBMCs.
4. Once the averages were determined, the percent of specific lysis for each condition was calculated using the following formula-
   
   
5. The percent of specific lysis was calculated for each condition (Table 2 - cells J4 to O4, for unstimulated PBMCs and J7 to O7, for unstimulated PBMCs).