Single-Cell Analysis

Antibody Selection and Preparation for Circulating
Tumor Cell (CTC) and Rare Cell Isolation
Using the Celselect Slides Enumeration Assay
Quick Guide

The Genesis System and Celselect Slides™ utilize microfluidics and individual microchambers to efficiently capture rare cells based on
their size. After capture, enriched cells can be recovered for downstream analysis or stained on-slide for immunofluorescent applications
such as enumeration and identification of various CTCs and other rare cell types using microscopy.
A critical step for successful enumeration is antibody panel design. The process involves the selection of suitable antibodies andsubsequent determination of the optimal antibody concentration to use.
This guide takes you through key considerations for creating an optimized staining panel, which is part of the sample preparation stepof the Celselect Slides Enumeration Direct or Indirect Stain Kits workflow (Figure 1).

Add fixing reagent to sample
and load sample onto
the instrument. Sample
and staining reagents areprepared according topredefined protocols.

Genesis System automates capture andimmunostaining of cells on Celselect Slides.

Image Celselect Slides witha fluorescent microscope in
the laboratory.

Import file from microscope toRare Cell Analysis Software,confirm cells of interest, andgenerate report.

Fig. 1. Celselect Slides Enumeration Direct or Indirect Stain Kits workflow.

Considerations for Antibody Selection and Preparation
1. Target Cell

First, clearly establish what type of cell is being enumerated.
There are two main cell types to choose from: epithelial andmesenchymal. Common examples of epithelial cell tumors include
breast, colon, and prostate cancers. Mesenchymal cells arecommonly seen in sarcomas.
■

E xample: MCF-7 cells are breast cancer cells, epithelial in origin,derived from adenocarcinoma of the mammary glands

2. Target Antigens

To identify your target cell type, you need to determine which
expressed antigens will enable positive identification. Antigens canbe expressed on the target cell surface or intracellularly, but you

Visit bio-rad.com/url for more information.

should also consider which antigens are not expressed by yourtarget cell, as often a negative result can also be used to confirm
identity. It is important to note that antigens are not mutually
exclusive to one specific cell type. Therefore, careful analysis of
antigen expression of the cell types present in your sample will beneeded to ensure that the targets chosen allow detection of thetarget cells only.
Bio-Rad lists the immunogen on the datasheets for each antibody,and provides a handy reference guide for human immune cellmarker expression. Immunogens are antigens that evoke immune
responses. Knowing the immunogen will allow for appropriate
antibody selection and therefore target cell identification.
■

Example: for MCF-7 cells, the antigen target is cytokeratin (CK)

Antibody Selection and Preparation for Circulating Tumor Cell (CTC) and Rare Cell Isolation Using the Celselect Slides Enumeration Assay

3. Antibody Staining Method■

There are two options (Figure 2); in both cases, the antibody
binding to its target antigen is visualized by a fluorescent detection
system, where the fluorophore used will fluoresce at a specificwavelength (nm) when exposed to a specific light source.
■ irect — this method utilizes a fluorescently conjugated primaryD
antibody to bind to the target antigen— It is possible to use the same host species for multiple
antibodies if a different subisotype is available. Isotypes are
generalized into categories such as IgG, IgM, etc. If using thesame species, each antibody should have a subisotype of
IgG1 or IgG2b— Protocol: Celselect Slides CTC Enumeration Direct Staining
User Manual■Indirect — this method uses an unconjugated primary antibody
that binds to the target antigen, after which a fluorescently
conjugated secondary antibody binds to the primary antibody

An example panel is Bio-Rad’s Celselect Slides Enumeration
Indirect Stain Kit (#CEL80111), which uses Mouse Anti-Human
CD45 (#MCA87) with an IgG2a isotype in tandem with a Mouse
Anti-Human PanCK (#MCA6434GA) IgG1 isotype. Here, ananti-pan-IgG secondary antibody should not be used, as crossreactivity would occur.

— Protocol: Celselect Slides CTC Enumeration Indirect Staining
User Manual
As more than one fluorescently conjugated secondary
antibody can bind to a single primary antibody, the fluorescentsignal can be significantly amplified compared to the direct
staining method.
A

Indirect staining — similarly, the primary antibody must be
specific for the target species (human cells) and antigen. It
is important to note that when using two or more primary
antibodies in one indirect staining panel, the isotypes and/or
host species should be unique to prevent cross-reactivity ofthe secondary antibody

5. Secondary Antibody Selection (Indirect staining only)

The isotype of the primary antibody needs to be the target
antigen for the secondary antibody. For example, Bio-Rad’s
CD45 antibody (#MCA87) has an isotype of IgG2a; therefore, thesecondary antibody must detect the IgG2a (Goat Anti-Mouse IgG2a[#STAR133] would be a suitable secondary antibody in this case).
The conjugated format of the secondary antibody will need to becarefully selected. Refer to consideration 6, where fluorophore
selection is discussed in detail.

B

6. Fluorophore Selection

For successful target cell visualization, the fluorophore needs to be
selected carefully.
■

Fig. 2. Diagram of direct and indirect staining. A, direct staining — a fluorescently
conjugated primary antibody binds directly to its target antigen. B, indirect staining— three fluorescently conjugated secondary antibodies indirectly detect a target
antigen by binding a primary antibody bound to the antigen.

■

4. Primary Antibody Selection

Bio-Rad recommends that monoclonal antibodies be used for
Celselect Slides Assays, as they are highly specific for an individual
region of the target antigen. This is not the case with polyclonal
antibodies, which detect multiple epitopes (which may lead tocross-reactivity to those same epitopes expressed on related
proteins), resulting in higher background signal. Primary antibody
selection depends on which staining method is used:■■ irect staining — the primary antibody must be specific for theDtarget species (human cells) and antigen. The conjugated formatof the primary antibody will need to be carefully selected; refer toconsideration 6, where fluorophore selection is discussed in detail© 2023 Bio-Rad Laboratories, Inc.

2 nderstand the configuration of the fluorescent microscopeUbeing used, in order to match the fluorophores you choose to
the filters available to detect them elect bright fluorophores that are highly photostable and have aS
low susceptibility to photobleaching. Conventional fluorophores
such as fluorescein isothiocyanate (FITC) and R-phycoerythrin(RPE) are especially prone to photobleaching, which results inquick fading of the fluorescent signal. To reduce photobleaching,use fluorophores with high photostability
 hen more than one secondary antibody is being used, it isW
important that the fluorophores selected fluoresce at different
wavelengths so that each cell population can be identified
individually. If the emission spectra of two fluorophores overlap,
one fluorophore might spill/bleed into another fluorophore filter.
Bleed-through can make it difficult or sometimes impossible todistinguish the two fluorophores. When selecting fluorophores,
the emission and excitation spectra should therefore bechecked for spectral overlap with the help of a fluorescence
spectra viewer. Ideally, there should be no overlap between the
fluorophore used in your immunofluorescence (IF) experiment.
Bio-Rad has a tool that shows the emission spectra offluorophores (bio-rad-antibodies.com/spectraviewer.html)

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Antibody Selection and Preparation for Circulating Tumor Cell (CTC) and Rare Cell Isolation Using the Celselect Slides Enumeration Assay■ fter selecting the fluorophore for your experiment, you mustA
decide which antigen to detect with which fluorophore. The
brightest fluorophore should be reserved for the detection of
the antigen with the least abundant expression level. The
dimmest fluorophore should be used for detecting the mostabundant antigen

Table 1. Assay setup for antibody titration.
Slide
Primary
Number Antibody

The optimal concentration of the antibody (primary and, if relevant,secondary antibody) will need to be determined to avoid highlevels of nonspecific (off-target) background staining from excess
antibody or loss of signal intensity from too little antibody, both ofwhich can prevent visualization of your target cell. Each antibody
will need to be titrated to determine the appropriate concentration
for target cell identification.

Always include positive and negative controls in every
experiment. Remember that the antibodies for the negative
controls will need to be titrated.

3.

When determining the optimum antibody concentration, run
the experiment with a representative cell line that is known toexpress the target antigen.

4.
5.

2

■

IgG2a Isotype
Detection antibody
Negative Control for CD45 antibody(#MCA929)(DyLight 650-conjugated
IgG2a antibody■ OR no primary
antibody added [#HCA310D650])

Rule out falsepositive staining
by the secondary
antibody alone3■

IgG1 Isotype
Detection antibody for
Negative Control ■ PanCK antibody(#MCA1209)(FITC-conjugated IgG1
antibody [#STAR132F])■ IgG2a Isotype

Rule out crosstalk
between thetwo secondary
antibodies■

OR no primary
antibody added■

4

Key Points

2.

■

Negative Control(#MCA929)

Antibody Titration and Experiment Setup
As a starting point for the titration, the recommended dilution for
immunofluorescence stated on the product datasheet provided
by the antibody supplier can be used. If a recommended dilution
is not available, the starting dilution for the titration of the
Celselect Slides Assay can be determined by performing a titrationin an IF experiment. During the process of determining the
optimum antibody concentration and experimental setup, you may
need up to six Celselect Slides plus a further two Celselect Slides
to run the assay.
Only one concentration of the titration can be performed at
any one time. Begin with the recommended starting dilution.

IgG1 Isotype
Detection antibody
Negative Control for the PanCK antibody(#MCA1209)(FITC-conjugated IgG1
antibody [#STAR132F])■ OR no primary

Purpose1

Rule out falsepositive staining
by the secondary
antibody alone

antibody added

7. Optimal Primary and Secondary Antibody Concentration

1.

Secondary
Antibody

5

6

Repeat with an antibody at a different dilution if the signal is
too high or too low.
Run the Direct or Indirect Enumeration Protocol for eachantibody being tested. Ensure you follow the guidance exactly,or the results will be invalid.

Anti-PanCK
antibody(#MCA6434GA)

Anti-CD45
antibody(#MCA87)■

Anti-PanCK
antibody(#MCA6434GA)■

Anti-CD45
antibody(#MCA87)

CD45 antibody(DyLight 650-conjugated
IgG2a antibody[#HCA310D650])

Detection antibody
for PanCK antibody(FITC-conjugated IgG1
antibody [#STAR132F])

Detection antibody
for CD45 antibody(DyLight 650-conjugated
IgG2a antibody[#HCA310D650])

Detection antibody for■ PanCK antibody(FITC-conjugated IgG1
antibody [#STAR132F])■

CD45 antibody(DyLight 650-conjugated
IgG2a antibody[#HCA310D650])■

Determine optimal
concentration
for primaryand secondary
antibodies■

Confirm signal
detected withoutbackground signal
from the antiCD45 antibody■

Determine optimal
concentration
for primaryand secondary
antibodies■

Confirm signal
detected withoutbackground signal
from the antiPanCK antibody

Confirm panels workon representativecell lines known
to express target
antigen

FITC, fluorescein isothiocyanate; IgG, immunoglobulin G.

Note that these antibodies can be tested in IF first to determine thebest primary and secondary antibody concentrations, to give an
optimal starting concentration.

Table 1 outlines an example titration and assay setup for thedetection of PanCK-positive cells (expressed on CTCs) using the
Celselect Indirect Enumeration Protocol.

After each slide assay finishes, analyze the slide with a fluorescent
microscope of choice. Bio-Rad recommends Agilent’s BioTek
Lionheart LX Automated Microscope with BioTeK Gen5 V3.14
Software, which has protocols that can be used to scan CelselectSlides. Furthermore, Bio-Rad’s Rare Cell Analysis Software iscompatible with the scanned images from this system.

© 2023 Bio-Rad Laboratories, Inc.

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Antibody Selection and Preparation for Circulating Tumor Cell (CTC) and Rare Cell Isolation Using the Celselect Slides Enumeration Assay

After obtaining the images, Rare Cell Analysis Software can beused to determine target cell counts. To use this software, ensure
all image files are one of the following file types: TIFF, JPEG, PNG,
or BMP.

Once the slides have been analyzed on a fluorescent microscope,
determine the target cell counts for each slide. If they arecomparable, the antibody panel has been optimized and can beused for further testing on this sample and target cell type, using
these antibodies at the optimized concentration. It should be notedthat new cell and sample types (even if also epithelial in origin) willneed separate antibody optimization.

If background fluorescence is observed for an antibody (Figure 3),
the gain or integration time may be too high.
A

B

Figure 4 shows the ideal antibody staining result using Bio-Rad’s
Standard Indirect Enumeration Panel:
■

■

C

■

Fig. 3. Representative images of MCF-7
cells stained with anti-PanCK antibody
and acquired at different parameters.
A, optimized scanning parameters
showing clear staining pattern of MCF-7
cells with balanced brightness andcontrast. B, high level of background signal
due increased exposure time, integration
time, and camera gain. C, image acquired
using low exposure time, resulting in dim
fluorescence signal that limits visualizationof cells of interest. Scale bar = 200 μm.

To reduce the background, try lowering the gain and integration
time parameters and rescanning before adjusting the antibody
concentration. If lowering these parameters does not eliminate
the background fluorescence, titrate the antibody down to a lower
concentration and then retry scanning.

 uclear Marker — 4',6-diamidino-2-phenylindole (DAPI) (blue)Nshould be present, indicating that a cell is present in the
Celselect Slides chamber. Note that DAPI is already in chambernumber 10 in the cartridge as part of the kit
 TC Marker — PanCK detected by FITC should be present,C
indicating detection of target cell type
 hite Blood Cell Marker — CD45 detected by DyLight 650
Wshould not be present, confirming that the cell detected is not acontaminating white blood cell

A

B

C

D

If no background fluorescence is noted for each single antibody
tested, run the panel of antibodies in duplicate against the cellline of interest in whole blood, as per the example assay (Table 2).
Table 2. Final assay confirmation setup.
Slide
NumberPrimary
Antibody

7■Anti-PanCK
antibody(#MCA6434GA)■

Anti-CD45
antibody(#MCA87)8■

Anti-PanCK
antibody(#MCA6434GA)■

Anti-CD45
antibody(#MCA87)

Secondary
Antibody

Purpose

Detection antibody for■ PanCK antibody(FITC-conjugated IgG1
antibody [#STAR132F])

Confirm panels
work on the cellline of interest(MCF-7 cells)■CD45 antibody(DyLight 650-conjugated
IgG2a antibody[#HCA310D650])

Detection antibody for■ PanCK antibody(FITC-conjugated IgG1
antibody [#STAR132F])■

E

CD45 antibody(DyLight 650-conjugated
IgG2a antibody[#HCA310D650])

Replicate slide to
double-confirm
that the panels
work on the cellline of interest(MCF-7 cells)

Fig. 4. Representative images of Celselect Slides CTC Staining Panel.
MCF-7 target cells spiked in human whole blood are identified using CTC marker(anti-PanCK antibody) and detected by FITC-conjugated IgG1 antibody. White blood
cells are identified using leukocyte marker (anti-CD45 antibody) and detected by
DyLight 650–conjugated IgG2a antibody. A, bright field image; B, DAPI image; C,
DyLight 650 image; D, FITC image; E, merged image. Scale bar = 200 μm. CTC,
circulating tumor cell; DAPI, 4',6-diamidino-2-phenylindole.

FITC, fluorescein isothiocyanate; IgG, immunoglobulin G.

© 2023 Bio-Rad Laboratories, Inc.

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Antibody Selection and Preparation for Circulating Tumor Cell (CTC) and Rare Cell Isolation Using the Celselect Slides Enumeration Assay

In Summary
Bio-Rad’s Celselect Slides Enumeration Assay is an effective
tool to identify target cell populations using size selection andantibody staining.
By ensuring that the antibody panel is optimized, users canbe confident that they are getting highly sensitive and specificidentification of the target cell population.
For further information on Bio-Rad’s Celselect Slides Enumeration
Panel and Rare Cell Analysis Software, see:■■

Celselect Slides products
Rare Cell Analysis Software Guide

Visit bio-rad-antibodies.com for more information.
BIO-RAD and CELSELECT SLIDES are trademarks of Bio-Rad Laboratories, Inc. in certain
jurisdictions. All trademarks used herein are the property of their respective owner.
© 2023 Bio-Rad Laboratories, Inc.

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