Application Note
Antibody validation with mRNA transfection into human pluripotent stem cells

visualization of primary antibodies the secondary
antibody Alexa Fluor 594 (1:500) was used.

Introduction
Elixirgen
Scientific’s
proprietarytranscription
factor-based technology allows rapid and reproducible
differentiation of human induced pluripotent stem cells(iPSCs) or embryonic stem cells (ESCs) into neurons
without sacrificing the purity of the cells1.
Immunocytochemistry (ICC) is used as one of the tools tocharacterize iPSC-derived neuron cells. Although
antibodies are one of the most commonly used tools inboth basic and clinical research, there are no universally
accepted standardized methods to validate reagents.
Three key aspects are involved in antibody validation:
specificity, affinity, and reproducibility. This applicationnote describes an easy method to validate antibody
specificity for immunocytochemistry using iPSCs.

Materials and methods
Cell culture and mRNA transfection
Human iPSCs (CW50065, CIRM iPSC repository) weremaintained with our standard protocols2. For eachantibody iPSCs were seeded at 25,000 viable cells perwell of a 96-well plate (36 wells) on day 0 and allowed togrow until the next day. Half of the wells (18 wells) werethen transfected with the mRNA encoding the target
protein, either ALDH1L1 or GFAP, using Lipofectamine™
MessengerMAX™ (ThermoFisher) as a positive control.
The remainder of the wells were left untransfected as anegative control. On day 2, the cells were fixed andstained with antibodies specific for the target proteins(Figure 1).

Figure 1: Schematic of the workflow for antibody validation.

Antibodies
Antibodies specific for GFAP and ALDH1L1 were used forvalidation. This method can be extended to otherantibodies that are not expressed in iPSCs. For

Immunocytochemistry
After fixing the cells with 4% paraformaldehyde, cells
were blocked with a blocking buffer. The cells transfectedwith mRNA were treated with three dilutions of antibodies(1:100, 1:200 and 1:500 for the GFAP antibody; 1:500,1:1000, and 1:2000 for the ALDH1L1 antibody) and threedifferent dilutions of IgG corresponding to theconcentration of primary antibodies. Untransfected cells
were treated with the same conditions. Samples wereprepared in triplicate. Primary antibody incubation wasdone in a blocking buffer overnight at 4℃ degrees. After 3
PBS
washes, cells were incubated in secondary
antibodies and nuclei were fluorescently stained with
Hoechst 33342.
Image acquisition and analysis
To capture immunofluorescence of a single well of a 96
well plate, 4 fields were imaged with a 10x objective using
an automated high-content confocal microscopy CQ1(Yokogawa). The nucleus and antibody positive areas
were segmented by an image analysis software (CellPathFinder, Yokogawa), and the number of total cells andof target antibody positive cells were counted from thenumber of nuclei segmented and from the mean intensityof target antibody fluorescence in segmented area,respectively.

Results
In order to validate antibody specificity, iPSCs transfected
and untransfected with target mRNA were treated withtarget antibodies or Immunoglobulin G (IgG) as anegative control (Figure 2A and 2B). Both transfected
cells treated with ALDH1L1 and those treated with GFAP
antibodies showed mosaic-like staining as thetransfection efficiency is not 100%. No signal wasobserved in untransfected cells treated with the target
antibody thus indicating no non-specific binding of theantibody. Transfected cells treated with IgG also showedno fluorescence indicating that this antibody is specific.
Captured images were analyzed by CellPathfinder. The
program identified stained nucleus and cytoplasmic
areas and quantified the mean intensity of fluorescence.
Figure 3A shows images of transfected andLast revised: November xx, 2022

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1untransfected cells treated with ALDH1L1 antibody andthe resulting identification/segmentation. Individual cells
can be segmented by specifying nuclear and cell regions.
The total cell number was counted. More than 10,000
cells were counted per well and the mean intensity offluorescence was plotted on the histogram (Figure 3B).
The IgG treatment resulted in no fluorescence.

Figure 2: Fluorescence images of iPSCs treated with ALDH1L1(A) and GFAP (B) on day 2. Blue: Hoechst 33342, Red:
ALDH1L1 or GFAP. Scale bar: 100µm.

Although it was not obvious in the images, somebackground staining was detected with the ALDH1L1antibody in untransfected iPS cells by CellPathfinder. The
profile of transfected cells treated with ALDH1L1 showed
two peaks. When a threshold was set based on
untransfected cells treated with ALDH1L1, it waspositioned in a valley and showed 72% positive cells intransfected cells with ALDH1L1 while the rest of theelixirgensci.com

samples showed 0.1% positive cells. We categorized thisantibody as “Fair”. With GFAP, peaks of all negative
controls indicated that there was no non-specific bindingor background fluorescence in untransfected cells. Athreshold was set just after the peaks of all negativecontrols and transfected cells treated with GFAP showedabout 66% positive cells. We categorized this antibody as“Good”. A dilution of primary antibody that showed
minimum background and non-specific binding waschosen for further use. If any of the negative controls
show more than 5% positive cells, we categorize theantibody as “Bad” and will not use it further.

Figure 3: Nucleus area and red color positive area segmented
by an image analysis software, Cell Pathfinder (A). Light blue
circles mean nuclei area and yellow circles mean red
fluorescent positive area. Scale bar means 100µm.
Histograms of mean red intensity in the nucleus area detectedby the image analysis (B). If both negative control samples,
untransfected cells stained with the target antibody andtransfected cells stained with IgG, it means that the target
antibody has very low non-specific binding.

2

Using the validated antibodies in this study, Quick-Glia™
Astrocytes
Human
iPSC-derived
astrocytes(AS-SeV-CW50065-S,Elixirgen
Scientific)
wereevaluated. The astrocytes were fixed at day 43 from
iPSCs and stained by GFAP and ALDH1L1 antibodies.
Figure 4 shows representative images of ICC. Using IgG
as a negative control, we determined the threshold for
GFAP positive cells. On the other hand, as the antibody

validation result suggested that ALDH1L1 had aunspecific binding, the threshold was not determinedfrom IgG negative control result, but from a profile ofhistogram of ALDH1L1 fluorescent intensity. As a result,the percentage of GFAP positive cells and that ofALDH1L1 were 50% and 94%, respectively.

Figure 4: Fluorescence images of iPSC-derived astrocytes stained with astrocyte markers GFAP and ALDH1L1 (A) Nuclei were stainedwith Hoechst 33342.. Scale bar = 100µm. (B) The histograms of mean green and red intensity in the nucleus area were calculated. From
the antibody validation experiment, the threshold of GFAP positive cells can be determined from IgG negative control. On the other hand,as the ALDH1L1 antibody showed some unspecific signal, the threshold was determined from the profile of the histogram.

Discussion and Summary

The variability of antibody quality has made validationof antibodies crucial. We have described a method tovalidate antibody specificity by transfecting iPSC withmRNA for the antibody targets. Using the CQ1 foranalysis allows this method to provide quantitative
data that can be used to set thresholds to categorize
antibodies and determine whether they should beused in future studies.

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References
1. Goparaju, S. K. et al. Rapid differentiation ofhuman pluripotent stem cells into functional
neurons by mRNAs encoding transcriptionfactors. Sci. Rep. 7, 1–12 (2017).
2. Application protocol of Elixirgen Scientific,
Maintenance of hPSC culture suitable for
Quick-Tissue™ Differentiation Kits

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