What is the recommended starting confluence to observe staining?

Name: GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit
Brand: R&D Systems
SKU: SC023B

The optimal confluence would depend on the purpose of the staining. If the plan is to use all the cells in the flask after they have proliferated, then one can wait for the cells to become confluent or almost confluent (80-90% confluent) and then check the staining. If the goal is to to check on the cells mid-way through their growth and there are sufficient cells in the flask, one could check the staining then as well. The staining is not dependent on how many cells are in the flask, as long as there are sufficient cells.

GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit (SC023B) by R&D Systems, Part of Bio-Techne

Kit Summary

To verify pluripotency in live, unfixed human stem cells and continue in culture.

Key Benefits

Verify human stem cell pluripotency in 30 minutes
Includes 4 pluripotent stem cell-specific fluorochrome-conjugated antibodies
Single-step protocol
No adverse effects on stemness or proliferation
Pick pluripotent human stem cell colonies with increased confidence

SC023B Data Examples
View Data Examples

Stemness vs. Pluripotency

Why Analyze Human Stem Cell Pluripotency by Live Cell Staining Using Stem Cell Markers?

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.

Designed to verify pluripotency in live cells, the GloLIVE^™ Human Pluripotent Stem Cell Live Cell Imaging Kit increases research efficiency and success by allowing single-step staining of unfixed cells.

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 Contents

This kit contains the following azide-free, fluorochrome-conjugated antibodies to verify human stem cell pluripotency:

Positive Markers

NL493-conjugated Mouse Anti-Human SSEA-4
NL557-conjugated Mouse Anti-Human TRA-1-60(R)
NL493-conjugated Mouse Anti-Human TRA-1-81

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

Stability and Storage

Reagents are stable for 12 months from date of receipt when stored in the dark at 2^° C to 8^°.

Data Examples

Human Induced Pluripotent Stem Cells Express TRA-1-81.
JOY1 human induced pluripotent stem cells were stained with the NL493-conjugated TRA-1-81 (green) antibody included in the GloLIVE^™ Human Induced Pluripotent Stem Cell Live Cell Imaging Kit. Cells were counterstained with Hoechst 33342 (blue).

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. 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.

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).

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Product Specifications for GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit

Species	Human
Source	N/A

Scientific Data Examples for GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit

	Human Induced Pluripotent Stem Cells Express TRA-1-81. JOY1 human induced pluripotent stem cells were stained with the NL493-conjugated TRA-1-81 (green) antibody included in the GloLIVE^™Human Induced Pluripotent Stem Cell Live Cell Imaging Kit. Cells were counterstained with Hoechst 33342 (blue).
	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.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.
	Analysis of Human Embryonic Stem Cell Growth and Stemness Post-staining. 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).

Preparation & Storage

Shipping Conditions	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage	Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human stem cell pluripotency can be verified in live cells prior to colony selection or experimentation using this 30 minute procedure:

Pluripotent stem cell marker antibodies are added directly to the cells of interest
After 30 minutes, the cells are washed and analyzed for the expression of pluripotency markers
Positive colonies can be selected for expansion in culture

View Graphic Procedure

Reagents Provided

Reagents supplied in the GloLIVE^™ Human Pluripotent Stem Cell Live Cell Imaging Kit (Catalog # SC023B):

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)
NL493-conjugated Mouse Anti-Human TRA-1-81 (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.

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GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit

R&D Systems, part of Bio-Techne | Catalog # SC023B

Key Product Details