An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells
JoVE Encyclopedia of Experiments
Immunology
0 views • 3:54 min • July 8th, 2025
Take a suspension of Jurkat cells — T lymphocyte leukemia cells.
Mix an aliquot with a cell viability dye and ensure an adequate number of living cells.
Plate the cells in a culture dish and apply ultraviolet C or UVC, radiation for the required duration.
UVC penetrates the cells and is absorbed by DNA, forming pyrimidine photoproducts.
Post-irradiation, incubate the cells. Extensive UVC-mediated DNA damage initiates the intrinsic apoptosis pathway, activating pro-apoptotic proteins on the outer mitochondrial membrane.
Activated proteins oligomerize, forming membrane pores and releasing cytochrome C from the intermembrane space into the cytoplasm.
Cytochrome C complexes with the apoptotic protease activating factor-1, forming an apoptosome complex. The apoptosome activates caspases, causing apoptosis.
Introduce fluorophore-labeled annexin V to label phosphatidylserine on the membrane, an apoptotic cell marker, and add propidium iodide that enters through the damaged membrane to label the DNA.
Using flow cytometry, determine the annexin V-labeled early apoptotic cells and dual-stained late apoptotic cells.
Begin by culturing Jurkat T cells in basal cell culture medium, supplemented according to the manuscript directions, at 37 degrees Celsius and 5% carbon dioxide. Jurkat T cells are a suspension cell line that can be maintained through passaging into pre-warmed culturing media every three days.
Prepare apoptotic cells by growing them to 90% confluency, which takes three to four days after passaging. Grow cells in five T75 flasks, to obtain a sufficient number of cells for this protocol. Once the cells are ready, aspirate the flask contents about 24 milliliters, and transfer them to a 50-milliliter conical tube. Count the cells using trypan blue staining and a hemocytometer.
Pellet the cells by centrifugation at 271 times g for five minutes, and discard the supernatant. Then, resuspend the cells in media to obtain three million cells per milliliter. Aliquot the cells into 100-by-20 millimeter tissue culture dishes by transferring five milliliters of cells per dish.
Prepare approximately nine culture dishes. Set one dish aside as an unexposed control, and expose the remaining dishes to UV. Set the UV crosslinker to the correct energy level by pressing the energy button and entering "600" with the number pad. Irradiate all dishes with the cells except for the control, making sure to remove the top cover of the tissue culture dish during UV exposure.
Then, incubate all the dishes in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide for four hours. After the incubation, combine all the irradiated cells into a 50-milliliter conical tube, and pellet them by centrifugation at 271 times g for five minutes. Discard the supernatant, and resuspend the cells in 24 milliliters of sterile PBS. Centrifuge the tube again, and remove the supernatant.
Overview
This article details a protocol for inducing apoptosis in Jurkat T lymphocyte leukemia cells using ultraviolet C (UVC) radiation. The process involves assessing cell viability, exposing cells to UVC, and analyzing apoptotic markers through flow cytometry.
Key Study Components
Area of Science
- Cell biology
- Apoptosis
- Oncology
Background
- Jurkat cells are a widely used model for studying T cell leukemia.
- UVC radiation is known to cause DNA damage, leading to apoptosis.
- Understanding apoptosis mechanisms is crucial for cancer research.
- Flow cytometry allows for the analysis of apoptotic cells based on specific markers.
Purpose of Study
- To demonstrate a method for inducing and analyzing apoptosis in Jurkat cells.
- To explore the effects of UVC radiation on cell viability and apoptosis pathways.
- To provide a detailed protocol for researchers in the field.
Methods Used
- Jurkat cells are cultured and prepared for UVC exposure.
- Cells are irradiated with UVC to induce DNA damage.
- Post-irradiation, cells are incubated and analyzed for apoptosis.
- Flow cytometry is used to quantify early and late apoptotic cells.
Main Results
- UVC exposure leads to significant DNA damage in Jurkat cells.
- Apoptosis is confirmed through the activation of caspases and the formation of apoptosomes.
- Flow cytometry effectively distinguishes between early and late apoptotic cells.
- The protocol provides reproducible results for studying apoptosis.
Conclusions
- The study successfully demonstrates a reliable method for inducing apoptosis in Jurkat cells.
- UVC radiation is an effective tool for studying apoptotic pathways.
- This protocol can aid researchers in understanding cancer cell biology.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables reproducible generation of apoptotic T cells for mechanistic studies in immuno-oncology and immunotherapy development. By providing a standardized method to induce apoptosis via UVC-mediated DNA damage, it supports target validation and pathway interrogation in T cell leukemia models. The approach enhances predictive confidence in preclinical screening by supplying well-characterized apoptotic cells for downstream assays such as efferocytosis or phagocytosis screening.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of apoptotic pathways in Jurkat T cells to validate targets involved in programmed cell death.
- Operational Value: Provides a consistent source of apoptotic cells for mechanistic de-risking of apoptosis-modulating therapeutics.
Screening & Assay Development
- Scientific Value: Generates apoptotic cells with defined phosphatidylserine exposure for use in efferocytosis or phagocytosis assays.
- Operational Value: Supports assay standardization through reproducible induction of early and late apoptosis stages quantifiable by flow cytometry.
Translational & Preclinical Research
- Scientific Value: Supports translational biomarker studies by enabling analysis of apoptotic cell clearance mechanisms relevant to autoimmune and inflammatory disease models.
- Operational Value: Facilitates preclinical evaluation of compounds targeting apoptotic signaling or phagocytic receptors using a standardized apoptotic cell substrate.
Pipeline & Workflow Integration
This method fits within the discovery continuum from target validation through assay development to preclinical evaluation, particularly in immuno-oncology and inflammation research where apoptotic cell function is a key readout.
- Discovery Biology: Supports hypothesis testing of apoptotic regulators and pathway clarification in T cell leukemia models.
- Screening: Enables preparation of apoptotic cells for high-content or flow-based screening of phagocytic or immunomodulatory compounds.
- Analytics: Provides quantitative readouts via flow cytometry using annexin V and propidium iodide to distinguish apoptotic stages.
- Translational Research: Connects to preclinical continuity by modeling apoptotic cell interactions in disease-relevant systems such as alveolar macrophage efferocytosis.
- Enterprise Reuse: Establishes a reusable platform for generating apoptotic T cells across multiple projects studying cell death, clearance, or immune tolerance.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in apoptosis-related target validation through standardized, reproducible cell preparation.
- Operational Value: Enhances reproducibility and scalability of apoptotic cell generation for multi-user laboratory environments.
- Strategic Value: Reduces biological variability in apoptosis assays, improving go/no-go decision reliability in early-stage programs.
- Portfolio Impact: Enables risk-adjusted prioritization of therapeutics targeting apoptotic pathways or phagocytic clearance mechanisms.
Implementation Considerations
- Requires expertise in cell culture, UV irradiation safety, and flow cytometry analysis.
- Dependent on access to a UV crosslinker with adjustable energy settings and flow cytometers capable of annexin V/PI detection.
- Necessitates standardization of irradiation duration, cell density, and incubation times across users and sites.
- Adaptation to primary T cells or other lymphocyte lines may require optimization of UV dose and viability thresholds.
- Limited to in vitro apoptosis induction; does not model in vivo microenvironmental influences on cell death.
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Last updated: 29 August 2026