GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit Summary
Kit Summary
Why Analyze Human Stem Cell Pluripotency by Live Cell Staining Using Stem Cell Markers?
Designed to verify pluripotency in live cells, this kit increases research efficiency and success by allowing single-step staining of unfixed cells. See Details
Maintaining pluripotent human embryonic stem (ES) cells and deriving induced pluripotent stem (iPS) cells requires colony selection and cell expansion. Manually picking colonies that contain pluripotent stem cells can be achieved by analyzing colony morphology. However, colony morphology is not truly indicative of pluripotency, since cells can naturally differentiate within the colony. Moreover, colony picking is a time-consuming and labor-intensive process.
Live pluripotent stem cell imaging:
- Promotes the selection of high quality, undifferentiated stem cell colonies to reduce experimental variation.
- Verifies stem cell pluripotency in 30 minutes, ensuring efficient use of time and reagents.
- Provides increased confidence in pluripotent status through the use of positive and negative markers.
- Allows for the continued culture of pluripotent cells with no adverse effects on cell proliferation or stemness following staining.
Kit Components
This kit contains the following azide-free, fluorochrome-conjugated antibodies to verify human stem cell pluripotency: See Details
Positive Markers
- NL493-conjugated Mouse Anti-Human SSEA-4
- NL557-conjugated Mouse Anti-Human TRA-1-60(R)
Negative Marker
- NL557-conjugated Mouse Anti-Human SSEA-1
Each antibody is supplied as a 50X stock; enough for 25 assays when used in 500 µL staining volume per assay. Each 1X antibody solution has an endotoxin level of < 5 EU/mL.
Data Examples
 View Larger Image | Verification of Pluripotency in Human Induced Pluripotent Stem Cells. iPS2 human induced pluripotent stem cells were grown on irradiated mouse embryonic fibroblasts (Catalog # PSC001) and stained using antibodies included in the Human Pluripotent Stem Cell Live Cell Imaging Kit (Catalog # SC023). A. iPS2 cells stained with the NL493-conjugated SSEA-4 (green) and NL557-conjugated SSEA-1 (red) antibodies. B. iPS2 cells stained with the NL493-conjugated SSEA-4 (green) and NL557-conjugated TRA-1-60(R) (red) antibodies. The cells were counterstained with Hoechst 33342 (blue). The colonies are positive for the stem cell markers SSEA-4 and TRA-1-60(R) and are negative for SSEA-1, suggesting that these colonies primarily contain undifferentiated human stem cells. |
 View Larger Image | Analysis of Human Embryonic Stem Cell Growth and Stemness Post-staining. A. BG01V human embryonic stem cells were plated in triplicate in 6 well plates at 0.5 x 106 cells per well on Day 0. On Day 1, cells were stained with (green bars) or without (blue bars) 1X NL-conjugated antibody. On Day 4, cells were harvested and counted using a hemocytometer. There was no effect of SSEA-4, TRA-1-60(R), or SSEA-1 staining on cell proliferation. Error bars indicate standard deviation. B. BG01V human embryonic stem cells were stained with (green histogram) or without (blue histogram) 1X NL-conjugated antibody and cultured for 3 days. Cells were then harvested and stained using the pluripotent marker PE-conjugated Rat Anti-Human/Mouse Oct-3/4 Monoclonal Antibody (Catalog # IC1759P; open histogram) or a Rat IgG2B PE-conjugated Isotype Control (Catalog # IC013P; filled histogram). Staining with SSEA-4, TRA-1-60(R), or SSEA-1 had no effect on stemness as analyzed by Oct-3/4 expression. Stemness post-staining was also verified by SSEA-4 and SSEA-1 expression (data not shown). |
BG01V human embryonic stem cells are licensed from ViaCyte, Inc.
Specifications
Product Datasheets
Assay Procedure
Refer to the product datasheet for complete product details.
Reagents Provided
Reagents supplied in the Human Pluripotent Stem Cell Live Cell Imaging Kit (Catalog # SC023):
- NL557-conjugated Mouse Anti-Human SSEA-1 (negative marker)
- NL493-conjugated Mouse Anti-Human SSEA-4 (positive marker)
- NL557-conjugated Mouse Anti-Human TRA-1-60(R) (positive marker)
Note: These antibodies have been tested for immunocytochemistry using human induced pluripotent stem cells grown either on irradiated mouse embryonic fibroblast feeder cells (Catalog # PSC001) or in feeder-free conditions. Each antibody is supplied as a 50X stock; enough for 25 assays when used in 500 µL staining volume per assay.
Other Supplies Required
Reagents
- Stem cell culture media
Materials
- Human pluripotent stem cells
- Pipettes and pipette tips
- Serological pipettes
Equipment
- 37 °C and 5% CO2 incubator
- Fluorescence microscope
Procedure Overview
To ensure sterility of cultures, all steps should be performed under sterile conditions.
Note: Culture with antibodies does not affect cell proliferation or stemness as assayed by proliferation curves and expression levels of SSEA-4, SSEA-1, and Oct-3/4 three days post-antibody incubation.
- Add primary antibodies in appropriate culture media to cells.
- Incubate for 30 minutes.
- Replace with fresh culture media.
- Visualize using a fluorescence microscope.
Citation for GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Comparative roadmaps of reprogramming and oncogenic transformation identify Bcl11b and Atoh8 as broad regulators of cellular plasticity
Authors: A Huyghe, G Furlan, J Schroeder, E Cascales, A Trajkova, M Ruel, F Stüder, M Larcombe, YB Yang Sun, F Mugnier, L De Matteo, A Baygin, J Wang, Y Yu, N Rama, B Gibert, J Kielbassa, L Tonon, P Wajda, N Gadot, M Brevet, M Siouda, P Mulligan, R Dante, P Liu, H Gronemeyer, M Mendoza-Pa, JM Polo, F Lavial
Nature Cell Biology, 2022-09-08;24(9):1350-1363. 2022-09-08
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