An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells
Published: February 29, 2024
Abstract
Source: Simonetti, S., et al. A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice. J. Vis. Exp. (2021)
This video demonstrates an assay to analyze antigen-specific CD8+ T cells in different cell cycle stages. Fluorescently-labeled viable cells are selected using flow cytometry, and the cells in the different cell cycle stages are identified by their DNA content using antibodies against cell proliferation-associated nuclear protein and a nucleic acid stain.
Protocol
1. Preparation of medium and staining solution Prepare Complete Medium: Roswell Park Memorial Institute (RPMI) medium with 2 mM glutamine, 100 U/mL penicillin/streptomycin, 50 µM beta-mercaptoethanol, and 10% volume/volume (v/v) of fetal bovine serum (FBS) Prepare Staining Buffer: Phosphate-buffered saline without Ca2+/Mg2+ (PBS) with 1% weight/volume (w/v) bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid disodium salt (EDTA) …
Representative Results
Figure 1: Cell cycle analysis of BM cells. BM cells from untreated Balb/c mice were stained and analyzed by flow cytometry. (A) Example of gating strategy. We gated on single cells in the DNA-A/-W plot (left) and subsequently on live cells by dead cell dye exclusion (middle). Then, a "relaxed" FSC-A/SSC-A gate was used for all BM cells (right). (B) Example of cell cycle analysis of BM cel…
開示
The authors have nothing to disclose.
Materials
| 1-200 μL universal fit bulk packed pipet tips | Corning | CLS4866-1000EA | |
| 2.4G2 anti-FcγR mAb | BD | 553141 | 10 μg/ml final concentration |
| 5 ml syringe plunger | BD Emerald | 307733 | |
| 15 ml conical tubes | MercK Millipore | SBHA025SB | |
| 60 mm TC-treated Cell Culture Dish | Falcon | 353002 | |
| 70 μm cell strainer | Falcon | 352097 | |
| 96-well Clear Round Bottom TC-treated Culture Microplate | Falcon | 353077 | |
| Anti-Rat/Hamster Ig,k/Negative Control Compensation Particles | BD- Bioscience | 552845 | |
| Beta-mercaptoethanol | Sigma | M3148 | |
| Bovine Serum Albumin | Sigma | A07030 | |
| BUV805 Rat Anti-Mouse CD8a | BD- Bioscience | 564920 | 4 μg/ml final concentration |
| Dulbecco's Phosphate Buffer Saline w/o Calcium w/o Magnesium | Euroclone | ECB4004L | |
| Eppendorf Safe-Lock Tubes, 1.5 mL | Eppendorf | 30120159 | |
| Ethanol | Sigma | 34852-1L-M | |
| Ethylenediaminetetraacetic Acid Disodium Salt solution (EDTA) | Sigma | E7889 | |
| Fetal Bovine Serum | Corning | 35-079-CV | |
| Filcon, Sterile, Syringe-Type 70 μm | Falcon | 352350 | |
| Fixable Viability Dye eFluor 780 | eBioscience | 65-0865-14 | 1:1000 final concentration |
| Foxp3 / Transcription Factor Staining Buffer Set | eBioscience | 00-5523-00 | This Set contains fixation/permeabilization concentrate and diluent, and permeabilization buffer 10x |
| H-2k(d) AMQMLKETI allophycocyanin (APC)-labelled tetramer | provided by NIH Tetramer Core Facility | 6 μg/ml final concentration | |
| H-2k(d) AMQMLKETI phycoerythrine (PE) labelled pentamer | Proimmune | F176-2A-E – 176 | 10 μL / sample |
| Hoechst 33342, Trihydrochloride, Trihydrate – 10 mg/mL Solution in Water | ThermoFisher | H3570 | |
| Ki-67 Monoclonal Antibody (SolA15), FITC | eBioscience | 11-5698-82 | 5 μg/ml final concentration |
| L-Glutamine 100X (200 mM) | Euroclone | ECB3000D | |
| Millex-HA Filters 0,45 µm | BD | 340606 | |
| Penicillin/Streptomycin 100X | Euroclone | ECB3001D | |
| PE/Cyanine7 anti-mouse CD62L Antibody | Biolegend | 104418 | 0.2 μg/ml final concentration |
| PerCP-Cy™5.5 Hamster Anti-Mouse CD3e | BD- Bioscience | 551163 | 4.4 μg/ml final concentration |
| Red Blood Cell Lysis Buffer | Sigma | R7757 | |
| Round-Bottom Polystyrene Tubes, 5 mL | Falcon | 352058 | |
| RPMI 1640 Medium without L-Glutamine with Phenol Red | Euroclone | ECB9006L | |
| Software package for analyzing flow cytometry data | FlowJo | v.10 | |
| Software for acquisition of samples at flowcytometer | BD FACSDiva | v 6.2 | |
| Trypan Blue Solution | Euroclone | ECM0990D |
