Flow cytometry Anti-bodies Human - B7-H4

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

B7-H4 Monoclonal Antibody (188), PE, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
	cells were incubated on ice for 30min with appropriate antibodies or isotype controls, washed twice with FACS buffer (PBS containing 0.1% NaN3 and 5% fetal bovine serum), and resuspended in 0.5 ml 1% formalin/PBS. To analyze intracellular B7-H4, cells were pre-treated with Fix and Perm cell permeabilization reagents (Caltag Laboratories) according to the manufacturer's instructions.	All analyses were performed on a FACS calibur system (Becton Dickinson Immunocytometry Systems).

Protocol tips
cells were incubated on ice for 30min with appropriate antibodies or isotype controls, washed twice with FACS buffer (PBS containing 0.1% NaN3 and 5% fetal bovine serum), and resuspended in 0.5 ml 1% formalin/PBS. To analyze intracellular B7-H4, cells were pre-treated with Fix and Perm cell permeabilization reagents (Caltag Laboratories) according to the manufacturer's instructions.
Downstream tips
All analyses were performed on a FACS calibur system (Becton Dickinson Immunocytometry Systems).

Manufacturer protocol Publication protocol 1 Relevant paper

APC Mouse Anti-Human B7-H4

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Each cell sample (1 × 106) was incubated with the APC-conjugated B7-H4 protein for 30 min at 4 °C. The cells were washed twice and were analyzed by flow cytometry.

Protocol tips
Each cell sample (1 × 106) was incubated with the APC-conjugated B7-H4 protein for 30 min at 4 °C. The cells were washed twice and were analyzed by flow cytometry.

Manufacturer protocol Publication protocol 1 Relevant paper

PE Mouse anti-Human B7-H4

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Fluorescence-activated cell sorting (FACS) analysis was performed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ). The following antibodies were used to classify the cells: anti-CD3-PE-Cy5, anti-CD14-FITC, anti-HLA-DE-PE, anti-B7-H1-PE and anti-B7-H4-PE (BD PharMingen, Franklin Lakes, NJ).

Protocol tips
Fluorescence-activated cell sorting (FACS) analysis was performed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ). The following antibodies were used to classify the cells: anti-CD3-PE-Cy5, anti-CD14-FITC, anti-HLA-DE-PE, anti-B7-H1-PE and anti-B7-H4-PE (BD PharMingen, Franklin Lakes, NJ).

Manufacturer protocol Publication protocol 1 Relevant paper

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