Flow cytometry Anti-bodies Mouse - CD117/c-kit

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 3 matching solutions for this experiment

Upstream tips
Samples were isolated from mouse tissues or tumors as above or from trypsinized from tissue culture samples.
Protocol tips
Unstained samples and samples with antibody IgG controls were run in parallel. Additionally, color compensation for each fluorochrome was evaluated using IgG compensation beads (552843, BD Pharmingen) and ArC compensation beads (A10346, Invitrogen) with appropriate fluorescent marker controls.
Downstream tips
Flow cytometry was performed in the Stanford Flow Cytometry Core Facility using the BD Aria II (sorting) or BD LSR.II (analysis).
Protocol tips
For c-kit+/SSEA1− cell isolation, cells were detached using HyQTase (Thermo Fisher Scientific), blocked with Fc (BioLegend, San Diego, CA, USA) for 15 min, and then incubated with the antibody at 4 °C for 30 min. Rinsing with staining buffer (eBioscience, San Diego, CA, USA).
Downstream tips
The cells were purified using a FACSAria Flow Cytometer (BD Bioscience). The purified cells were passaged five times before differentiation assays and cell transplantation.
Protocol tips
Cultured cells were analyzed with FACS using anti-mouse CD19, CD3, and c-kit antibodies (BD Biosciences).
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms