Purified Mouse Anti-Human CD33

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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
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.
Downstream tips
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.
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