An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells

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…

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