Chromium Release Assay: The Old School Way of Testing Cytotoxic T Cells

There are several methods you can use to see if your T cells are cytotoxic, but a chromium release assay using radioactive ⁵¹chromium (⁵¹Cr) is one of the oldest. It gives good results, and is great for labs that can’t afford or don’t have flow cytometry readily available.

Here, I will outline a simple method for testing T cell killing function using ⁵¹Cr. But before you start your assay, be sure to read up on proper safety measures when handling radioactive materials.

Pick Your Target and Effectors

To do a chromium release assay, you first incubate your target cells (T) with ⁵¹Cr. You then incubate the target cells with effector cells (your T cells; E). If the effector cells kill the targets, then they will release the ⁵¹Cr, which you will detect with a gamma counter.

Which targets and effectors you use is entirely dependent on your application. Your target cells are what you are testing T cell function against. You might want to kill your target (measure cytotoxicity) or you could be looking to see if the T cells are tolerant (like testing manufactured cells before they go into a patient for safety!). Your effector T cells can also be derived from different sources. For example, you can use manufactured T cell lines or T cells freshly isolated from an animal.

Let’s Get Radioactive

Once you’ve gathered your targets and effectors, it’s time to get cooking.

1. Count your cells. You can use a hemocytometer or an automatic cell counter. The number of cells you need is dependent upon the number of conditions and how many replicates of each condition you do. Typically, I run my assays in triplicate and have four ratios (5:1, 10:1, 20:1 and 40:1 E:T). Aliquot your target cells (in my case 500,000- about 25,000 per well) in to 15ml conical tubes, keeping each experimental condition separate! Make sure you suspend your cells in a small volume so they have the chance to come in contact with the chromium.

2. Add the ⁵¹Cr (around 50 ?Ci) and incubate your targets with the chromium for one hour. Flick the tubes about every 20 minutes to increase opportunities for cells to uptake the chromium.

3. While you’re incubating….This long incubation is a great time to set up your effector cells. Wash the T cells two times with PBS to remove excess cytokines that could skew your results. Then resuspend them in the appropriate media.

4. Once you have finished the washes, you’re ready to put your effectors on the plate! I typically use a 96 well plate since the volumes are rather small. Plate the effectors in different ratios to the targets to determine the threshold of killing. I typically run the assay in triplicates but it depends on how many cells you have available. 40, 20, 10, and 5 to 1 ratios (Effectors to Targets) are a good template if you have enough available cells.

In addition to these test, set up two control conditions:

• a spontaneous release control containing just target cells (basically a negative control because there should be no killing without the effectors) and
• a maximum release control in which you lyse the target cells (a positive control that shows the maximum amount a chromium taken up by the cells).

5. Back to those Target cells…Once your target cells are done incubating, wash them three times with cell culture medium to remove the excess chromium. Spin the cells gently at 400g for 5 min for each of these washes. Decant the supernatant into a waste container that can be transferred to a safe decay area rather than trying to pipet it out. This will help reduce possible contamination since you won’t have to deal with any tips.

6. After the washes, resuspend your cells to 5000 cells per 100?l. Then add 100?l of cells to each well, even the spontaneous and maximum wells. Incubate the cells together for a minimum of 4 hours at 37°C, exactly how long depends on your application.

7. Four hours later….Take your plate out of the incubator. Add 100?l of a 1% triton-x solution to your cells in the maximum wells to lyse the cells and release all the chromium.

8. Spin the plate down at 1250rpm for ten minutes to pellet the cells.

9. Transfer the supernatant from each well to the well of an absorbent plate (e.g. Luma plates) being careful not to touch the pellet at the bottom of the well.

10. Leave the plate to dry overnight. In the morning, pop it into the plate reader and get your results.

The results you see will depend on your specific application

Calculating the Results of Your Chromium Release Assay

Once you have the readings, you need to perform a simple calculation to determine the cytotoxicity of your cells.

First, average your triplicates for each condition. Then take those averages and plug them into this formula:

(Chromium release for condition of interest – Chromium release in spontaneous wells)/ (max chromium release – Chromium release in spontaneous wells)

Multiply the value by 100 to get your percent lysis.

Would you use a chromium release assay? Have you already? Comment below!