Immunology JoVE (Open Access) Citable · DOI

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Name: Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2
Uploaded: 2026-06-04
Duration: 12 min 43 s
Description: Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all ...

DOI: 10.3791/50856-v

What you'll learn

✓ Perform FACS-based enrichment of human embryonic stem cells using TG30 and GCTM-2 surface markers
✓ Apply positive and negative selection strategies to isolate pluripotent hESC populations
✓ Culture sorted cell subfractions and assess pluripotency after immunofluorescent sorting

Protocol

We describe the use of the monoclonal antibodies TG30 (CD9) and GCTM-2 for the combined detection of cell surface antigens via fluorescence activated cell sorting (FACS) for the identification and enrichment of live human embryonic stem cells (hESC) using positive selection and also the use of negative selection to purge hESCs from a mixed cell population.

Difficulty

advanced

Total time

~4–6 hours per sample (excluding culture recovery periods)

Model organism

Human embryonic stem cells (hESC)

Biosafety

BSL-1

Steps

Dissociate hESC bulk cultures into single cells

Enzymatically digest human embryonic stem cell cultures to generate single-cell suspensions suitable for flow cytometry analysis and sorting.

▶ 01:38

Perform immunofluorescent staining for TG30 and GCTM-2

Incubate dissociated cells with monoclonal antibodies TG30 (CD9) and GCTM-2 to label cell surface antigens prior to FACS analysis and sorting.

▶ 02:44

Culture sorted cell subfractions post-FACS

Plate enriched or depleted hESC populations from FACS sorting into culture vessels and maintain under standard pluripotency-supporting conditions.

▶ 05:02

Enrich pluripotent hESC from TG30Hi-GCTM-2Hi gates

Identify and collect high-expressing double-positive cells via positive selection in FACS to establish enriched pluripotent hESC populations for downstream analysis.

▶ 07:46

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