Xiaohuan Wang1, Ming Lei1, Peifang Ye1,
Yan Lu1, Garret Guenther2, Nan Li2

Autofluorescence subtraction by
spectral flow cytometry

CYTO 2024#46

1Agilent Biosciences (Hangzhou) Co., Ltd.,

China, 2Agilent Technologies, Inc., USA

Introduction

Results and Discussions

Cells have a natural level of fluorescence, called
autofluorescence (AF), which is caused by thepresence of fluorescent biological compounds within
the cells. AF can result in the loss of signal resolution
and the presence of false positive events.

Autofluorescence subtraction from mouse lung cells

AB

Spectral flow cytometers can treat autofluorescencesignal as a background or a “virtual” fluorochrome andextract it from the signal of fluorophore-labeled cells.
The removal of AF can improve the unmixing
accuracy and the signal resolution of fluorochromesin samples consisting of high AF signals.
Herein we compare the unmixed results with orwithout the subtraction of various AF across different
samples. These samples contain highly homogenousor heterogeneous AF properties, including humanblood and mouse lung cells.

C Unstained sample
Without AF subtraction

With AF subtraction

ExperimentalMouse lung sample preparation
Mouse lungs were extensively perfused with HBSS
through a right ventricle, cut into small pieces, anddigested with enzymes. Homogenized lung cells werepassed through 70-μm nylon mesh and red blood
cells were lysed using RBC lysing buffer. Following
that, cells were collected and stained by mixed
antibodies in the table below. Dead cells were stainedwith Live/Dead Blue.
Specificity
CD45
Sca-1
EpCAM（CD326）
CD11b
Ly6G
Siglec-F
F4/80
CD11c
CD103
MHC IICD64
CD24
Ly6C
Live/DeadClone
30-F11
D7
G8.8
M1/70
1A8
E50-2440
BM8
N418
2E7
M5/114.15.2
X54-5/7.1
M1/69
HK1.4
NA

Fluorochrome
PerCP
APC-Fire750
eFluor450
PE-Cy7
BV605
PE-CF594
Kiravia Blue520
BUV805
PE
BV785
APC
BV510
BV711
Live/Dead Blue

SourceBiolegend
Biolegend
Thermo fisher
Biolegend
BiolegendBD
BiolegendBD
Biolegend
Biolegend
Biolegend
Biolegend
Biolegend
Thermo fisher

Catalog number
10312910814648-5791-82
101215127639562757123162749038121405107645139306101831
128037
L34961

Human blood sample preparation• CD-Chex Plus (stabilized human blood in apreservative medium, CAT#213323, Streck) werestained by mixed antibodies, CD45 Brilliant Violet
510, CD3 PerCP-Cy5.5, CD4 Brilliant Violet 421,
CD8 Pacific Blue, CD16CD56 PE, and CD20 FITC.
Subsequently, red blood cells were lysed.
Afterward, the samples were washed andresuspended.

D Full-stained sample
Without AF subtraction

With AF subtraction

E

1.Endothelial cells

2.Ⅱ Epithelial cells
5.Eos4.Neu•

Fresh human blood was lysed and washedwithout staining.
All samples were analyzed on a five-laser NovoCyteOpteon flow cytometer. All plots and spectra weregenerated by the NovoExpress (Opteon) software.

6.CD11b+ DC
3.AM

7.CD103+ DC

Results and Discussions
The autofluorescence spectra of different samples vary

11.IMA

10.Ly6C++
Mo/MФ

8.Ly6C- 9.Ly6C+
Mo/MФ Mo/MФ

Figure 2. Autofluorescence subtraction from mouse
lung
cells
with
highandheterogeneous
autofluorescence populations enables accurate
fluorescent unmixing and improved fluorochrome
resolution in a 14-color immunophenotyping panel. (A)The selection of unstained sample and AF tags isconvenient and flexible in the fluorochrome setting
panel of the NovoExpress (Opteon) softwareinterface. (B) is the spectra of ten AF tags identified
from an unstained mouse lung sample. Unmixing
results of (C) an unstained sample and (D) a fullstained sample with or without AF spectra
subtraction are compared. The aqua blue in (C) and(D) highlights a population of which the unmixing
without AF subtraction is inaccurate. (E) is the
manual gating strategy used to identify the mouselung cell subsets, including (1) Endothelial cells, (2) Ⅱ
Epithelial cells, (3) Alveolar Macrophages, (4)Neutrophils, (5) Eosinophils, (6) CD11b+ DendriticCells, (7) CD103+ Dendritic Cells, (8)Ly6CMonocyte/Macrophages,(9)
Ly6C+Monocyte/Macrophages,(10)
Ly6C++
Monocyte/Macrophages
and(11)
Interstitial
Macrophages.

Unmixed results with or without AF subtraction in a 6C Panel

B

Without AF subtraction

C
With AF subtraction

Figure 1. Density spectrum plots of unstained
populations from different samples. The AFs of (A)lymphocytes from fresh human blood and (B)lymphocytes from stabilized human blood are
homogenous, but the stabilized cells have muchhigher AF signals than those from fresh blood. There
are heterogeneous and strong AF signals in (C)mouse lung cells.

Conclusions•There is a high autofluorescent signal diversity inflow cytometry samples, not only in signal
intensity but also in spectrum signature.

•The removal of autofluorescence can improve thesignal resolution and unmixing accuracy.

•The combination of the NovoCyte Opteon spectral
flow cytometer and NovoExpress (Opteon)
software enables convenient and accurate
autofluorescence subtraction.

References
Figure 3. Autofluorescence extraction from stabilized
human blood with high homogenous autofluorescencesignals significantly improves fluorochrome resolution ina 6-color immunophenotyping panel.

[1] Surre, J., Saint-Ruf, C., Collin, V. et al. Strong increase in theautofluorescence of cells signals struggle for survival. Sci Rep 8,12088 (2018).

For Research Use Only. Not for use in diagnostic
procedures. RA # RA45411.4460763889