Reagents for single-step immunocytochemical staining of live unfixed human pluripotent stem cells.
R&D Systems™ GloLIVE Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC023B) contains four stem cell marker antibodies conjugated to NorthernLights™ (NL) fluorochromes that can be used for single-step, quick, direct immunocytochemical staining of live unfixed human pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells). The cells are able to continue in culture after staining without affecting cell proliferation or stemness.
- NL493-conjugated Mouse Anti-Human SSEA-4 Antibody
- NL557-conjugated Mouse Anti-Human TRA-1-60(R) Antibody
- NL493-conjugated Mouse Anti-Human TRA-1-81 Antibody
- NL557-conjugated Mouse Anti-Human SSEA-1 Antibody
Immunocytochemistry Procedure for this Kit:
These antibodies have been tested for immunocytochemistry (ICC) using human induced pluripotent stem cells grown either on irradiated mouse embryonic fibroblast (iMEF) feeder cells 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.
To ensure sterility of cultures, all steps should be performed under sterile conditions.
- Dilute the desired antibody to a 1X concentration (1:50) in appropriate culture media.
- Remove the media from the cells. Gently add fresh media containing 1X antibody, and return the cells to the incubator for 30 minutes.
- Remove the antibody containing media, rinse cells once with fresh media, and re-feed with fresh media.
- Cells can be visualized at this point or continued for further culture if desired.
Note: Culture with antibodies does not appear to affect cell proliferation or stemness.
Assessment of Marker Expression in Live Unfixed Stem Cells
|Figure 1. A. and B. iPS2 human induced pluripotent stem cells grown on irradiated mouse embryonic fibroblasts (Catalog # PSC001) were stained using antibodies included in the Human Pluripotent Stem Cell Live Cell Imaging Kit (Catalog # SC023B). A. NL493-conjugated SSEA-4 (green) and NL557-conjugated SSEA-1 (red). B. NL493-conjugated SSEA-4 (green) and NL557-conjugated TRA-1-60(R) (red). The nuclei were counterstained with HO-33342 (blue). C. BG01V human embryonic stem cells were plated in triplicate at 0.5 x 106 cells on Day 0. On Day 1, cells were stained with (pink bars) or without (red bars) 1X NL-conjugated antibody. On Day 4, cells were harvested and counted. There was no effect of SSEA-4, TRA-1-60(R), or SSEA-1 staining on cell proliferation. Error bars indicate standard deviation. D. BG01V human embryonic stem cells were stained with (green histogram) or without (red histogram) 1X NL-conjugated antibody and cultured for 3 days. Cells were then harvested and stained for 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).