Cells were detached with Versene (Invitrogen) counted and resuspended in 100 µL phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). Cells were blocked with PBS/1% BSA for 1 h on ice. Two micrograms of QUAD 3.0, EphA3 antibody (house made),9 (link) EphA2, EphB2 (R&D Systems), or IL-13RA2 (Biolegend) was added and incubated for 2 h on ice with occasional mixing. Cells incubated with isotype antibody served as controls. Cells were washed with PBS/1% BSA prior to the addition of Alexa-Fluor-labeled secondary antibodies. Cells were incubated on ice for an additional 1 h with occasional mixing. After washing with PBS/1% BSA, cells were postfixed with 10% buffered formalin. Cells were analyzed on an Accuri 6 flow cytometer. Data were analyzed using the FCS Express software (DeNovo Software).
Protocol detail
Antibody Staining and Flow Cytometric Analysis
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Antibodies
Antibody
Antibody isotype
Bovine serum albumin
Cells
Epha3
Ephb2
Formalin
Phosphate
Saline
Versene
Corresponding Organization : Atrium Health Wake Forest Baptist
Other organizations : Children's Hospital of Philadelphia, Wake Forest University, Virginia–Maryland College of Veterinary Medicine
Top 5 similar protocols
Variable analysis
independent variables
- Incubation time (2 h)
- Antibodies used (QUAD 3.0, EphA3, EphA2, EphB2, IL-13RA2, isotype control)
- Cell surface expression of the antibody targets
- Cell suspension (100 µL phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA))
- Cell blocking (PBS/1% BSA for 1 h on ice)
- Washing steps (PBS/1% BSA)
- Cell fixation (10% buffered formalin)
- Flow cytometry analysis (Accuri 6 flow cytometer, FCS Express software)
- Cells incubated with specific antibodies (QUAD 3.0, EphA3, EphA2, EphB2, IL-13RA2)
- Cells incubated with isotype antibody
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!