Flow cytometry Anti-bodies Mouse - CD45

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).

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Found 3 matching solutions for this experiment

Protocol tips
For cell-surface antigen staining, the samples were incubated with antibodies for 20 min at 4 °C, washed and resuspended in PBS containing 3% FBS. To exclude dead cells, 7-amino actinomycin D (7-AAD, BD Biosciences) was added to the samples before sorting.
Downstream tips
We sorted live, single cells using a FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences).
Upstream tips
Transfer 10 μl of the cell suspension to a 5-ml FACS tube containing 190 μl WM and leave on ice. This is the unstained control sample.
Protocol tips
Add 190 μl WM into each tube and 10 μl of the cell suspension. Add PI so that the final concentration is 0.3 μg/ml, add 0.25 μl CD45-APC
Upstream tips
Add 10 μl cell suspension from the 2 ml sort sample in 500 μl media and the following volumes of antibody to separate 5 ml FACS tubes for single color controls
Protocol tips
1 μl biotin CD45
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