Flow cytometry Anti-bodies Human - CD41

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

APC Mouse Anti-Human CD41a

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads.	Ab or appropriate isotype controls (all from BD Biosciences) were incubated for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark.	The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter). The number of BFU-E or CFU-E cells in one million MNCs was quantitated.

Upstream tips
To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads.
Protocol tips
Ab or appropriate isotype controls (all from BD Biosciences) were incubated for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark.
Downstream tips
The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter). The number of BFU-E or CFU-E cells in one million MNCs was quantitated.

Manufacturer protocol Publication protocol 1 Relevant paper

PE-Cy™7 Mouse Anti-Human CD41a

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	To measure microparticles, plasma aliquots were centrifuged for 2 min at 10,000xg to pellet residual cells and debris. The supernatant was collected and then centrifuged for 45 min at 17,000xg. The resulting microparticle pellet was resuspended in PBS containing 1% FCS and 2.5mM Ca+2 and incubated with Fc block for 10 min at 4°C.	After 30 min at room temperature, 25,000 volumetric counting beads were added and 10,000 events collected on an LSR II flow cytometer.

Protocol tips
To measure microparticles, plasma aliquots were centrifuged for 2 min at 10,000xg to pellet residual cells and debris. The supernatant was collected and then centrifuged for 45 min at 17,000xg. The resulting microparticle pellet was resuspended in PBS containing 1% FCS and 2.5mM Ca+2 and incubated with Fc block for 10 min at 4°C.
Downstream tips
After 30 min at room temperature, 25,000 volumetric counting beads were added and 10,000 events collected on an LSR II flow cytometer.

Manufacturer protocol Publication protocol 1 Relevant paper

CD41-PE, P2, 2 mL, ASR

Beckman Coulter

Upstream tips	Protocol tips	Downstream tips
For platelet staining, 100 μL of PRP was aliquoted into 12 × 75 mm round bottom tubes	The samples were incubated in a dark environment for 30 min at room temperature. After incubation, 500 μL of PBS was added to each tube and the samples were analysed on the BD FACSAria IIu cell sorter located in the CAF Fluorescence Microscopy Unit, Stellenbosch University. For each sample, a minimum of 30,000 events were acquired and all signal were gated.

Upstream tips
For platelet staining, 100 μL of PRP was aliquoted into 12 × 75 mm round bottom tubes
Protocol tips
The samples were incubated in a dark environment for 30 min at room temperature. After incubation, 500 μL of PBS was added to each tube and the samples were analysed on the BD FACSAria IIu cell sorter located in the CAF Fluorescence Microscopy Unit, Stellenbosch University. For each sample, a minimum of 30,000 events were acquired and all signal were gated.

Manufacturer protocol Publication protocol 1 Relevant paper

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