What is the recommended starting confluence to observe staining?

Name: GloLIVE Human TRA-1-60(R) NL493 Live Cell Antibody
Brand: R&D Systems
SKU: NLLC4770G

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 TRA-1-60(R) NL493 Live Cell Antibody (NLLC4770G) by R&D Systems, Part of Bio-Techne

Kit Summary

To detect TRA-1-60(R) expression in live cells by immunocytochemical staining.

Why confirm marker expression in live cells before colony selection?

Maintaining pluripotent human embryonic stem cells and deriving induced pluripotent stem cells require colony selection and cell expansion. Picking colonies that contain pluripotent stem cells is typically achieved by manually analyzing colony morphology. Unfortunately, this critical process is time-consuming and labor-intensive.

In addition, morphology-based colony selection does not consistently guarantee pluripotent starting populations, since cells can fail to be fully reprogrammed or can naturally differentiate within a colony.

To address this need, R&D Systems offers GloLIVE™ antibodies that allow researchers to confirm stem cell marker expression in live cells before colony selection.

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.
Allows for the continued culture of pluripotent cells with no adverse effects on cell proliferation or stemness following staining.

Antibody Components

GloLIVE NL493-conjugated Mouse Anti-Human TRA-1-60(R) Monoclonal Antibody.
Supplied as 50X concentration of antibody in 0.5 mL PBS

Azide free and endotoxin tested (= 5 EU/mL), this antibody can be used for 12 tests, if a staining volume of 2 mL is used.

TRA-1-60 is a monoclonal antibody raised against a cell surface antigen of human embryonal carcinoma (EC) cells (1). The TRA-1-60 epitope is also found on human embryonic stem (ES) cells and primordial germ cells, and TRA-1-60 serves as a serum marker in patients with germ cell tumors (2‑4). Investigation into the identity of the TRA-1-60 epitope demonstrated that it is a carbohydrate carried by a cell surface, sialylated, keratan sulfate proteoglycan (5). Subsequent evidence implicated podocalyxin as a carrier for the TRA-1-60 epitope (6).

Product Specifications for GloLIVE Human TRA-1-60(R) NL493 Live Cell Antibody

Species	Human
Applications	Immunocytochemistry/ Immunofluorescence

Scientific Data Examples for GloLIVE Human TRA-1-60(R) NL493 Live Cell Antibody

	Detection of TRA-1-60(R) in Live BG01V Human Embryonic Stem Cells. The stem cell marker TRA-1-60(R) was visualized in live BG01V human embryonic stem cells using a GloLIVE NL493-conjugated Mouse Anti-Human TRA-1-60(R) Monoclonal Antibody (Catalog # NLLC4770G; green). The nuclei were counterstained with Hoechst 33342 (blue). Positive staining for TRA-1-60(R) in human stem cells is an indicator of pluripotency.

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. Protect from light.

Background: TRA-1-60(R)

Item	Value
Long Name	Tumor-related Antigen-1-60 [R]
Entrez Gene IDs	5420 (Human)
Alternate Names	GCTM-2 antigen, GP135, PC, PCLP, PCLP-1, PDX, PODXL1, Podocalyxin, Podocalyxin-like protein 1, TRA-1-60(R), TRA160(R), gp200

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, TRA-1-60(R) expression can be verified in live cells prior to colony selection or experimentation using this simple procedure:

Add the TRA-1-60(R) antibody directly to the media of cultured cells
Wash the cells after a 30 minute incubation
Select for and expand TRA-1-60(R)-positive colonies

View Graphic Procedure

Precautions

Use aseptic technique and sterile culturing conditions to prevent contamination.

Reagents Provided

GloLIVE NL493-conjugated Mouse Anti-Human TRA-1-60(R) Monoclonal Antibody. (Catalog # NLLC4770G):

Other Supplies Required

Reagents

Stem cell culture media

Materials

Human or mouse pluripotent stem cells
Pipettes and pipette tips
Serological pipette

Equipment

37 °C and 5% CO₂ incubator
Fluorescence microscope

Live Cell Immunocytochemistry Procedure Overview

To ensure sterility of cultures, all steps should be performed under sterile conditions.

Add primary antibody in appropriate culture media to cells.

Add fluorochrome-conjugated primary antibody in appropriate culture media to cells.
Incubate for 30 minutes.

Replace with fresh culture media.

Visualize using a fluorescence microscope

Visualize using a fluorescence microscope.

Note: Culture with GloLIVE antibodies does not appear to affect cell proliferation or stemness as assayed by proliferation curves for 3 days post-antibody incubation and expression levels of TRA-1-60(R) and Oct-3/4.

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GloLIVE Human TRA-1-60(R) NL493 Live Cell Antibody

R&D Systems, part of Bio-Techne | Catalog # NLLC4770G

Key Product Details

Summary for GloLIVE Human TRA-1-60(R) NL493 Live Cell Antibody