Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

Summary

This protocol adapts cell cycle measurements for use in a mass cytometry platform. With the multi-parameter capabilities of mass cytometry, direct measurement of iodine incorporation allows identification of cells in S-phase while intracellular cycling markers enable characterization of each cell cycle state in a range of experimental conditions.

Abstract

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the cell cycle is a feature of a number of diseases, including cancer. Study of the cell cycle necessitates the ability to define the number of cells in each portion of cell cycle progression as well as to clearly delineate between each cell cycle phase. The advent of mass cytometry (MCM) provides tremendous potential for high throughput single cell analysis through direct measurements of elemental isotopes, and the development of a method to measure the cell cycle state by MCM further extends the utility of MCM. Here we describe a method that directly measures 5-iodo-2′-deoxyuridine (IdU), similar to 5-bromo-2´-deoxyuridine (BrdU), in an MCM system. Use of this IdU-based MCM provides several advantages. First, IdU is rapidly incorporated into DNA during its synthesis, allowing reliable measurement of cells in the S-phase with incubations as short as 10-15 minutes. Second, IdU is measured without the need for secondary antibodies or the need for DNA degradation. Third, IdU staining can be easily combined with measurement of cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated histone H3 (pHH3), which collectively provides clear delineation of the five cell cycle phases. Combination of these cell cycle markers with the high number of parameters possible with MCM allow combination with numerous other metrics.

Introduction

Mass cytometry enables detection of approximately 40 parameters by taking advantage of the high resolution and quantitative nature of mass spectroscopy. Metal-labeled antibodies are used instead of fluorophore conjugated antibodies that allow for a higher number of channels and produce minimal spillover^1,2. MCM has advantages and disadvantages in regard to cell cycle analysis in comparison to flow cytometry. One major advantage of MCM is that the large number of parameters enables the simultaneous measurement of cell cycle state across a large number of immunophenotypically distinct T-cell types in highly hete....

Protocol

1. Preparation of IdU stocks

1. Dissolve 5-iodo-2’-deoxyuridine (IdU) in DMSO to a concentration of 50 mM. Sterile filter, aliquot into 10-50 µL tubes, and store at -80 °C
2. Remove IdU from the freezer, and thaw at room temperature. Dilute IdU in RPMI-1640 to make a working solution at a final concentration of 1 mM. Pipette up and down or vortex to mix.
   1. Typically, dilute the concentrated IdU into the media in which the cells are being cultured (e.g. DMEM, IMDM, etc.) or have diluted it into PBS for addition directly to peripheral blood or bone marrow aspirate samples. This pre-dilution step facilitates mixing of the DMSO wi....

Results

Utilizing HL-60 cells and a human bone marrow aspirate it is possible to show how experimental conditions can affect cell cycle distribution and analysis. First, the gating strategy must be established to demonstrate how the cell cycle phases are derived. In Figure 1 we show the establishment of the singlet gate, which is important in separating cellular debris and doublets, establishing a single cell population. For cell lines the singlet gate is all that is needed to move onto cell cycle a.......

Discussion

The examples presented here demonstrate how to use an MCM platform to analyze cell cycle distribution. It has also been demonstrated that cell cycle analysis is sensitive to experimental conditions such as time and temperature, which is an important consideration researchers must take when considering MCM for their cell cycle analysis¹⁴. Samples left in storage for a short period of time, no longer than an hour, will have IdU incorporation comparable to their normal state. Samples in a closed syst.......

Materials

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA)	Sigma	A3059	Component of CSM
Centrifuge	Thermo Scientific	75-217-420	Sample centrifugation
Cleaved-PARP (D214)	BD Biosciences	F21-852	Identification of apoptotic cells
Cyclin B1	BD Biosciences	GNS-1	G2 Resolution
Dimethylsulfoxide (DMSO)	Sigma	D2650	Cryopreservative
EQ Four Element Calibration Beads	Fluidigm	201078	Internal metal standard for CyTOF performance
FACS Tube w/ mesh strainer	Corning	08-771-23	Cell strainer to remove clumps/debris before CyTOF run
Fetal Bovine Serum (FBS)	VWR	97068-085	Cell culture growth supplement
Helios	Fluidigm		CyTOF System/Platform
Heparin	Sigma	H3393	Staining additive to prevent non-specific staining
IdU (5-Iodo-2′-deoxyuridine)	Sigma	I7125	Incorporates in S-phase
Ki-67	eBiosciences	SolA15	Confirmation of G0/G1
MaxPar Multi Label Kit	Fluidigm	201300	Metal labeling kit, attaches metals to antibodies
Microplate Shaker	Thermo Scientific	88880023	Mixing samples during staining
Paraformaldehyde (PFA)	Electron Microscopy Services	15710	Fixative
pentamethylcyclopentadienyl-Ir(III)-dipyridophenazine	Fluidigm	201192	Cell identification during CyTOF acquisition
p-H2AX (S139)	Millipore	JBW301	Detection of DNA damage
p-HH3 (S28)	Biolegend	HTA28	M-phase Resolution
Phosphate Buffered Saline (PBS)	Gibco	14190-144	Wash solution for cell culture and component of fixative solution
p-Rb (S807/811)	BD Biosciences	J112906	G0/G1 Resolution
Proteomic Stabilizer	SmartTube Inc	PROT1	Sample fixative
RPMI 1640	Gibco	21870-076	Cell culture growth medium
Sodium Azide	Acros Organics	AC447810250	Component of CSM/Antibody buffer, biocide