Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Summary

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Abstract

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.

This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

Introduction

In the last few decades, flow cytometry (FCM) became a widely available analytical method essential for cell phenotyping studies. There has been a substantial increase in the available fluorescent dyes, particularly fluorochromes excited by the violet laser (405 nm) (e.g., Brilliant Violet and new Q-dot dyes). However, the growth of available fluorescent dyes increases the risk of overlapping emissions and requires labor-intensive compensation matrices. FCM became widely used to analyze cell suspensions from solid tissue, but the presence of auto-fluorescent cells limits the discrimination of specifically labeled populations.

The b....

Protocol

All experiments were performed according to the Pasteur Institute Ethic Charter and the EU guidelines and were approved by the French Agriculture Ministry.

1. Cell Suspension Preparation from Adult Mouse Organs

1. Isolation of splenocytes
   1. Euthanize adult mice by cervical dislocation. Make an incision at the abdomen midline and open the skin with scissors. Harvest the spleen with forceps.
   2. Crush the spleen between 2 glass microscope slides to dissociate the cells and dilute them in 5 mL of Hank's Balanced Salted Solution (HBSS) containing 1% fetal calf serum (FCS).
   3. Centrifuge for....

Results

21-parameter spectral FCM panel to analyze splenocytes

Figure 1 shows the results obtained with the 19-fluorescent-antibody panel applied to splenic cells comprising different antibodies recognizing subsets of T, B, NK, dendritic, and myeloid cells, while CD45 labels all hematopoietic nucleated cells. The panel also includes a viability dye (PI), as well as the size (FSC) and granularity (SSC) parameters. F.......

Discussion

Conventional FCM is based on the detection of photons emitted after the excitation of fluorescent probes. The fluorescence emission of one fluorochrome detected in a detector designed to measure the signal from another fluorochrome induces physical overlap. This spillover among emission spectra needs to be corrected by compensations.

Spectral FCM and data processing by the unmixing deconvolution algorithm allows for the combination of fluorochromes with close emission peaks without additional .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mice	Janvier Labs	CD45.2	Source of cells
Ethanol 70%	VWR	83801.36	Sterilization
DPBS (Ca2+, Mg2+)	GIBCO ThermoFisher	14040-174	Embryos collection
Hanks' Balanced Salt Solution (+Ca2+ +Mg2+) (HBSS+/+)	GIBCO Life ThermoFisher	14025092	Dissociation, digestion and staining solutions
Hanks' Balanced Salt Solution (-Ca2+, -Mg2+) (HBSS-/-)	GIBCO Life ThermoFisher	14175095	Dissociation, digestion and staining solutions
Fetal calf serum (FCS)	EUROBIO	CVFSVF00-0U	Dissociation, digestion and staining solutions
Collagenase	Sigma	C2139	Enzymatic solution
90 x 15 mm, plastic tissue culture Petri dishes.	TPP	T93100	Embryos and hearts collection
35 x 15 mm, plastic tissue culture Petri dishes.	TPP	T9340	Hearts collection
Fine iris scissor	Fine Science Tools	14090-09	Dissection tools
Gross forceps (narrow pattern forceps, curved 12 cm)	Fine Science Tools	11003-12	Dissection tools
Fine forceps (Dumont no. 7 forceps)	Fine Science Tools	11272-30	Dissection tools
Scalpel	VWR	21909-668	Dissection tools
LEICA MZ6 Dissection microscope	LEICA	MZ6 10445111	Sampling; Occular W-Pl10x/23
Cold lamp source	SCHOTT VWR	KL1500 compact	Sampling; Two goose neck fibers adapted
FACS tubes	VWR	60819-310	Digesting cells
96 well plate (round bottom)	VWR	10861-564	Staining cells
Nylon mesh bolting cloth sterilized, 50/50 mm pieces.	SEFAR NITEX	SEFAR NITEX	Cell filtration
UltrasComp eBeads
Fluorescence labeled antibodies	Biolegend	Catalogue number see table below	Staining cells