Pluripotent Stem Cell Verification: 3 Ways to Utilize a Single Well of a 24-well Plate

W. Johnson, G. Wegner, M. Andersen, S. Tousey, & J. Aho


Pluripotent stem cell verification is a crucial process both for newly established cell lines as well as during routine culture. Here, we describe three methods for easy stem cell verification using the small sample size of a 24-well plate:

  1. Verification by Antibody-Based Array: Membrane-based multiplex antibody array analysis verifies the expression of 15 different pluripotent and differentiated cell markers from the lysate of a single well of a 24-well plate, in less time than it takes to perform a single Western blot.
  2. Verification by Live Cell Imaging: GloLIVE™ live-cell imaging antibodies allow for quick verification of pluripotent marker expression in live cells within 30 minutes.
  3. Verification by Tri-lineage Differentiation: Directed tri-lineage differentiation functionally verifies cell potency within 5 days.

Together these tools assess cell quality with ease and reliability, leading to consistency among experiments while saving time, money, and reagents.


24 well plate

1. Verification by Antibody-Based Array

Verification by Antibody-Based Array
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BG01V human embryonic stem (ES) cells were differentiated in 24-well plates using the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). Lysate from a single well containing cells differentiated into endoderm, mesoderm, or ectoderm was added directly to an individual array from the Proteome ProfilerTM Human Pluripotent Stem Cell Array Kit (Catalog # ARY010). ES cell lysates (25 μg) were used as a positive control. A) Array data shows high expression of Oct-3/4 in undifferentiated ES cells which decreases upon tri-lineage differentiation. Differentiated cells increase protein expression for each of their respective cell-specific markers. B) Array data were confirmed by immunocytochemical staining. Undifferentiated ES cells were stained for Goat Anti-Human Oct-3/4 Polyclonal Antibody (Catalog # AF1759) while differentiated ES cells were stained for cell-specific markers: endoderm (Goat Anti-Human SOX17 Polyclonal Antibody; Catalog #AF1924), mesoderm (Goat Anti-Human Snail Polyclonal Antibody; Catalog #AF3639), and ectoderm (Goat Anti-Human Otx2 Polyclonal Antibody; Catalog #AF1979). Primary antibody staining was visualized with the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog #NL001). Each image was pseudo-colored to correspond with its respective label in A. The nuclei were counterstained with DAPI (blue).

2. Verification by Live Cell Imaging

Verification by Live Cell Imaging
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Live cell verification of BG01V human embryonic stem (ES) cell differentiation using SSEA-4 and SSEA-1 GloLIVE antibodies. ES cells were differentiated using the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). At specific time points during differentiation (days 0, 2, and 4) ES cells were stained with GloLIVE NL493-conjugated Mouse Anti-Human/Mouse SSEA-4 Monoclonal Antibody (green; Catalog # NLLC1435G) and GloLIVE NL557-conjugated Mouse Anti-Human/Mouse SSEA-1 Monoclonal Antibody (red; Catalog # NLLC2155R). As expected, ES cell differentiation was marked by the progressive loss of SSEA-4 staining and increased expression of SSEA-1. DAPI (blue) counterstain was used to visualize cell nuclei.

3. Verification by Tri-Lineage Differentiation

Verification by Tri-Lineage Differentiation
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One human induced pluripotent stem cell line (iBJ7) and one human ES cell line (BG01V) were differentiated into each of the three germ layers using the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). Differentiation was assessed by immunocytochemistry for endoderm (SOX17), mesoderm (Brachyury), and ectoderm (Otx2) using the primary antibodies included within the kit. The nuclei were counterstained with DAPI (blue).


  • Easily and routinely validate your stem cell populations with these three methods.
  • All three techniques can be performed on cells cultured and differentiated in a 24-well plate, limiting the number of cells needed for verification and conserving cells for experimentation.
  • Verification by antibody-based array, live cell imaging, and tri-lineage differentiation will ensure the highest quality of cells for downstream experiments, increasing result consistency and confidence.

For research use only. Not for use in diagnostic procedures.

BG01V human embryonic stem cells are licensed from ViaCyte, Inc.

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