Cd33 clone wm53

Manufactured by BioLegend

Sourced in Germany

CD33 (clone WM53) is a monoclonal antibody that binds to the CD33 antigen. CD33 is a transmembrane protein expressed on the surface of myeloid-derived cells, including monocytes, granulocytes, and some leukemia cells. This antibody can be used for the identification and characterization of CD33-positive cells in research applications.

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3 protocols using cd33 clone wm53

Multilineage Human Cell Engraftment

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Mice were bled via the tail vein to assess human cell reconstitution. Terminal mice were sacrificed with CO₂ followed by cardiac puncture for blood collection. BM cells were collected from the femurs. Blood samples and BM cells were incubated with ACK lysis buffer (Lonza Technologies) to lyse red blood cells. Leukocytes were resuspended in PBS, stained with the appropriate antibodies on ice for 20 minutes and washed. The following antibodies, specific for mouse CD45.1 (clone A20), human CD45 (clone HI30), CD13 (clone WM15), CD33 (clone WM53), CD38 (clone HIT2), CD47 (clone CC2C6), CD11b (clone M1/70), CD14 (clone M5E2), CD34 (clone 581), CD123 (clone 6H6), CD3 (clone HIT3a), CD56 (clone HCD56), CD19 (clone HIB19), CD45RA (clone HI100), and CD45RO (clone UCHL1), were purchased from BioLegend. Stained cells were resuspended in PBS + 10% FBS with DAPI and filtered. At least 10,000 events were collected on a LSRII flow cytometer (Becton-Dickenson). Data was analyzed with FlowJo software. Cell sorting was performed on an Aria3 machine (Becton-Dickenson). For secondary transplant experiments, sorted cells were resuspended in PBS for immediate tail vein injections. For mRNA processing, sorted cells were pelleted, snap-frozen and stored at −80°C.

Kaur M., Drake A.C., Hu G., Rudnick S., Chen Q., Phennicie R., Attar R., Nemeth J., Gaudet F, & Chen J. (2019). Induction and Therapeutic Targeting of Human NPM1c+ Myeloid Leukemia in the Presence of Autologous Immune System in Mice. Journal of immunology (Baltimore, Md. : 1950), 202(6), 1885-1894.

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Ex vivo Expanded AML Cell-NK Cell Co-culture

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Ex vivo expanded primary AML cells of 9 different patients were co-cultured with freshly isolated healthy donor NK cells, at an E:T ratio of 5:1 in an ex vivo long term culture system as described by Krupka and coworkers [29 (link), 54 (link)]. Antibodies were added at a final concentration of 10 nM. After 24 hours, cells were harvested, stained for CD16 (clone B73.1), CD56 (clone HCD 56), CD33 (clone WM53) and in concrete cases CD123 (clone 6H6; all antibodies from Biolegend) and analyzed by flow cytometry with a BD LSR II (Becton Dickinson, Heidelberg, Germany). The percentage of residual CD33 or CD123 positive cells in treated cultures relative to control cultures was used to determine licMAB-mediated cellular cytotoxicity. Patient characteristics are summarized in Supplementary Table S1.

Ponce L.P., Fenn N.C., Moritz N., Krupka C., Kozik J.H., Lauber K., Subklewe M, & Hopfner K.P. (2017). SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells. Oncotarget, 8(7), 11284-11301.

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Profiling Peripheral Blood Immune Cells

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Peripheral blood samples were collected before the first treatment cycle. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood on Ficoll-Hypaque gradients as previously described [23] (link). Fresh PBMCs from patients were stained with antibodies targeting CD4 (clone OKT4; Biolegend), CD8 (clone HIT8a; Biolegend), CD25 (clone BC96, Invitrogen), CD127 (clone ebioRDR5; Thermo Fisher), CD3 (clone UCHT1; Thermo Fisher), CD19 (clone HCD56; Biolegend) and CD56, CD33 (clone WM53, Biolegend), HLA-DR (clone LN3; Biolegend), CD14 (clone 61D3, Thermo Fisher), and CD15 (clone HI98; Biolegend). Intracellular staining for anti-human FoxP3 (clone PCH101; Thermo Fisher) was done after fixation and permeabilization with the FoxP3 staining set (Thermo Fisher).
Isotype controls were used as negative controls. Dead cells were eliminated using the Live/Dead Fixable Aqua Dead Cell Kit (Thermo Fisher). Stained cells were analyzed on a LSRII cytofluorometer using FACS Diva (Becton Dickinson) and Flow-Jo Software (TreeStar).

Pernot S., Terme M., Radosevic-Robin N., Castan F., Badoual C., Marcheteau E., Penault-Llorca F., Bouche O., Bennouna J., Francois E., Ghiringhelli F., De La Fouchardiere C., Samalin E., Baptiste Bachet J., Borg C., Boige V., Voron T., Stanbury T., Tartour E., Gourgou S., Malka D, & Taieb J. (2020). Infiltrating and peripheral immune cell analysis in advanced gastric cancer according to the Lauren classification and its prognostic significance. Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association, 23(1).

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