Flow cytometry Anti-bodies Human - CD71

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-H7 Mouse Anti-Human CD71

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

FITC Mouse Anti-Human CD71

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	and 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended.	The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired.

Protocol tips
and 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended.
Downstream tips
The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired.

Manufacturer protocol Publication protocol 1 Relevant paper

APC Mouse Anti-Human CD71

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Incubation 60 minutes at 4°C in staining buffer (2% BSA in PBS). Corresponding isotypes were used as control. Cells were washed in PBS, centrifuged, and finally resuspended in 300-μL analysis buffer (1% BSA/2 mmol/L EDTA in PBS).	The dead cells and debris were excluded after 7-AAD staining (Invitrogen). Data were collected on FACSAriaIII cell sorter (BD Biosciences) and analyzed using FlowJo software (TreeStar).

Protocol tips
Incubation 60 minutes at 4°C in staining buffer (2% BSA in PBS). Corresponding isotypes were used as control. Cells were washed in PBS, centrifuged, and finally resuspended in 300-μL analysis buffer (1% BSA/2 mmol/L EDTA in PBS).
Downstream tips
The dead cells and debris were excluded after 7-AAD staining (Invitrogen). Data were collected on FACSAriaIII cell sorter (BD Biosciences) and analyzed using FlowJo software (TreeStar).

Manufacturer protocol Publication protocol 1 Relevant paper

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