An equal number of cells were used for cell surface CD34 and/or CD11b staining together with the dead cell dye Zombie. In brief, DMSO, AURKA PROTACs, and ATRA (A9120, Solarbio) treated leukemia cells from the in vitro and in vivo assays were collected, counted, and washed with PBS. Subsequently, cells were resuspended and stained in a 100 µL staining buffer containing 1:100 diluted PE antihuman CD34 (343506, Biolegend) and/or 1:100 diluted Alexa Fluor 700 antimouse/human CD11b (101222, Biolegend) together with 1:500 diluted Zombie in the dark for 30 min at room temperature. After being washed with PBS, the cells were subjected to flow cytometry, while the relative cell surface CD34 and CD11b expression (MFI) were analyzed with CytExpert and FlowJo software.
Alexa fluor 700 antimouse human cd11b
Manufactured by BioLegend
Sourced in United States
Alexa Fluor 700 anti-mouse/human CD11b is a fluorescently-labeled antibody that binds to the CD11b protein, which is expressed on the surface of certain immune cells, such as monocytes, macrophages, and granulocytes. The Alexa Fluor 700 dye allows for detection and analysis of CD11b-expressing cells using flow cytometry.
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Lab products found in correlation
Fitc anti mouse cd3, by BioLegend (1 mentions)
Pe cy7 anti mouse cd19, by BioLegend (1 mentions)
Apc anti mouse cd8a, by BioLegend (1 mentions)
Zombie yellow fixable viability kit, by BioLegend (1 mentions)
Rat anti mouse cd16 cd32 antibody, by BD (1 mentions)
Pacific blue anti mouse cd4, by BioLegend (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Ab203919, by Abcam (1 mentions)
Aurka, by Cell Signaling Technology (1 mentions)
Pe antihuman cd34, by BioLegend (1 mentions)
α tubulin, by Beyotime (1 mentions)
Et1601 4, by Huabio (1 mentions)
C myc, by Beyotime (1 mentions)
3 protocols using alexa fluor 700 antimouse human cd11b
1
Quantification of CD34 and CD11b in Leukemia
2
Multiparametric Immune Cell Analysis
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Blood was centrifuged at 400 x g for 15 minutes. The blood pellet was repeatedly incubated in red blood cell lysis buffer (4.15 g NH4Cl, 0.55 g KHCO3, and 0.185 g EDTA disodium salt in 500 ml H2O) centrifuged until a white pellet was obtained. After adjusting the samples to 1 million cells per 100 μl PBS, the Fc receptors of immune cells were blocked with 10 μg/ml unlabeled purified rat anti-mouse CD16/CD32 antibody (#553142, BD Biosciences, Germany). Dead cells were stained with the Zombie Yellow™ Fixable Viability Kit (#423103, BioLegend, US). After centrifuging at 400 x g for 3 min, cells were incubated with 100 μl of an antibody mix containing FITC anti-mouse CD3 (#100203, BioLegend, US), APC anti-mouse CD8a (#100711, BioLegend, US), Pacific Blue anti-mouse CD4 (#100427, BioLegend, US), PE/Cy7 anti-mouse CD19 (#115519, BioLegend, US), APC/Cy7 anti-mouse NK-1.1 (#108723, BioLegend, US), Alexa Fluor700 anti-mouse/human CD11b (#101222, BioLegend, US), and a PE anti-mouse/rat/human CD27 (#124209, BioLegend, US) antibody. Unstained samples were used to control for autofluorescence during the flow cytometry measurements. Cells were measured by flow cytometry (BD LSR II FortessaT M SORP, BD Bioscience, Germany). The data were acquired with the BD FACSDiva™ Software and analyzed with FlowJo V10.5. The gating strategy is described in Figure S4 B.
3
Comprehensive Protein Expression Analysis
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Antibodies against GAPDH (ET1601‐4, Huabio), AURKA (14475S, Cell Signaling Technology), PARP (9532S, Cell Signaling Technology), c‐Myc (AF6513, Beyotime), pH3S10 (9701S, Cell Signaling Technology), H3 (4499, Cell Signaling Technology), Bcl‐2 (15071, Cell Signaling Technology), CRBN (AF6564, Beyotime), cIAP1 (GTX110087, GeneTex), VHL (68547, CST), cyclin E1 (AF2491, Beyotime), cyclin B1 (AF1606, Beyotime), STAT5A (AF2038, Beyotime), NANOG (ab203919, Abcam), FOXM1 (AF6924, Beyotime), AURKB (GTX132702, GeneTex), TACC3 (AF1345, Beyotime), TPX2 (GTX115654, GeneTex), Phospho‐TACC3 (Ser558) (AF4506, Affinity Biosciences), and Phospho‐Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (2914S, CST) were used for the Western blot analysis. PE antihuman CD34 (343506, Biolegend) and Alexa Fluor 700 antimouse/human CD11b (101222, Biolegend) were used for the flow cytometry analysis. AURKA (14475S, Cell Signaling Technology), α‐Tubulin (AT819, Beyotime), Alexa Fluor 488 goat antirabbit IgG (H+L) (A11034, Invitrogen), Alexa Fluor 546 goat antimouse IgG (H+L) (A11003, Invitrogen) were used for immunofluorescence assay.
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