FITC Mouse Anti-Human CD71

Protocol tips

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.

Publication protocol

"CD71 surface expression was measured on bone marrow blast cells. Staining to identify blastic cells was chosen based on the routine bone marrow immunophenotyping performed for diagnostic purposes. The antibodies (surface and cytoplasmic) included anti-CD3 (cytoplasmic), CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD20, CD22 (cytoplasmic), CD33, CD34, CD45, CD56, CD61, CD64, CD71, CD79a (cytoplasmic), CD117, GlyA (CD235a), HLA-DR, and MPO (cytoplasmic). All antibodies used were from Becton Dickinson (BD) Biosciences, San Jose, CA, USA. CD71 (FITC, clone L01.1) was used for this study. Company recommended amount of respective antibodies, 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.

The samples were then analyzed using a six-color flow cytometer (BD FACSVerse, Becton Biosciences, San Jose, CA, USA) with antibody panels against a variety of lymphoid, myelomonocytic, erythroid, and megakaryocytic antigens. List-mode data files were analyzed for each specimen using the FACSuite Software (Becton Biosciences, San Jose, CA, USA). Day-to-day fluorescence standardization of the flow cytometry was performed with BD Cytometer setup and tracking (CS&T) beads (Becton Biosciences, San Jose, CA, USA) by performing performance QC and assay and tube setting updates.

FACSuite software (Becton Biosciences, San Jose, CA, USA) was used for the analysis. A total of 20,000 cells were recorded per sample. Threshold gating was assigned based on forward scatter and side scatter (SSC) to exclude red blood cells, dead cells, and cell debris. A CD45/SSC gating strategy was used to gate the blasts cells. These cells were then plotted on CD71 histogram. The lymphocytes were taken as reference internal negative control. The staining intensity of CD71 on these cells was measured by the median fluorescence intensity (MFI). Because CD71 fluorescence and its respective control fluorescence were recorded using identical instrument settings, MFI values were independent of variations in signal amplification. CD71 was considered positive if the positive population comprised more than 20% of the cells. An unlabeled tube was run with each sample with only the gating marker (CD45) to set up the quadrant marker."

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