Flow cytometry Anti-bodies Mouse - CD49b

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

Upstream tips
At the indicated time points, tumor draining lymph nodes(tumor-DLNs) and spleens were harvested from the mice, and minced into small fragments and mechanically dispersed in 3-5 ml cold PBS. After filtering with 70 μm cell strainer (BD Falcon, USA), Single-cell suspensions were adjusted to 1 × 106 cells in 100 μl of PBS.
Protocol tips
After this, single-cell suspensions of tumor cells were stained for 30 min on ice with 1 μg of antibodies labeled with fluorochromes and then fixed and permeabilized with a permeabilization buffer (BD Biosciences, USA).
Downstream tips
analyzed by FACSCalibur (BD Biosciences, USA). Irrelevant IgG mAbs were used as a negative control. Ten thousands live events were acquired for analysis.
Protocol tips
Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell)
Downstream tips
Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences).
Protocol tips
cell were labeling in multiparameter flow cytometric analysis (FACS Calibur, BD Biosciences)
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