To examine surface CD11b expression, HL-60 cells after the treatment with and without DMSO were stained with APC-Cy™7 Mouse Anti-Human CD11b (557754, BD, San Diego, CA, USA). The cells were then subjected to flow cytometric analysis with a FACS Canto II (BD Biosciences, San Jose, CA, USA). Cells were analyzed using a gating strategy. An initial gate to isolate neutrophils was established based on the characteristics of FSC and SSC. CD11b expression of 10,000 gated neutrophils was determined as CD11b+ cells. Data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA) with 10,000 events per sample.
Apc cy 7 mouse anti human cd11b
Manufactured by BD
Sourced in United States
The APC-Cy™7 Mouse Anti-Human CD11b is a fluorochrome-conjugated monoclonal antibody that binds to the CD11b antigen expressed on the surface of human myeloid cells. It can be used in flow cytometry applications for the identification and analysis of myeloid cell populations.
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Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Hcs lipidtox red neutral lipid stain, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 700 mouse anti human cd19, by BD (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Perm buffer 3, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Pe mouse anti human cd86, by BD (1 mentions)
Apc mouse anti human cd34, by BD (1 mentions)
Pe cy7 mouse anti human cd14, by BD (1 mentions)
4 protocols using apc cy 7 mouse anti human cd11b
1
CD11b Expression in Differentiated HL-60 Cells
2
Immunophenotyping and Lipid Uptake Analysis
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Cells were fixed in 1% paraformaldehyde and when necessary, permeabilized with Perm Buffer III (558050, BD Biosciences, San Jose, CA), and blocked with FcR Blocking Reagent (130-059-901, Miltenyi Biotec, San Diego, CA). Antibodies used in flow cytometry analysis were: biotin mouse anti-human CD49e (555616, BD Biosciences) with streptavidin-APC (SA1005, Caltag, Buckingham, MK18 1TF), alexa fluor 647 mouse anti-fibronectin (563098, BD Biosciences), alexa fluor 700 mouse anti-human CD19 (557921, BD Biosciences), APC-Cy7 mouse anti-human CD11b (560914 BD Biosciences), PE mouse anti-human CD3 (9515-09, Southern Biotechnology, Birmingham, AL), APC mouse anti-human CD36 (561822, BD Biosciences). For lipid uptake analysis, HCS LipidTOX Red Neutral Lipid Stain (Invitrogen), Low Density Acetylated Lipoprotein from Human Plasma conjugated with Alexa Fluor 594 (Invitrogen). Cells were analyzed on the LSR II (BD Biosciences) or Accuri C6 (BD Biosciences) flow cytometers. Data were analyzed with FLOWJO software (Treestar, Ashland, OR, USA).
3
Flow Cytometric Analysis of M1 Macrophages
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M1 macrophages were detected by flow cytometry. In specific, the medium was discarded, and cells were washed with PBS two times, and then detached with 0.25% trypsin. After centrifugation, the precipitate was washed with PBS two times and the cells were counted. The cell concetration was adjusted to 1 × 106 cells/mL and samples then transferred into a 15 mL centrifuge tube along with 100 μL PBS containing 2% FBS. According to the kit instructions, the cells were added with specific fluorescent flow cytometry antibody APC-Cy™7 Mouse Anti-Human CD11b (560914, BD Bioscience) and PE Mouse Anti-Human CD86 (560957, BD Pharmingen) to identify M1 macrophages. The cells were incubated at 4 ℃ for 30 min in the dark, resuspended with 3 mL PBS, centrifuged, and added with 300 μL PBS buffer. In the control group, the background marker was determined by homotype monoclonal antibody. The fluorescence cells were analyzed by flow cytometry (BD FACSVerse, Becton Dickinson, Franklin Lakes, New Jersey, USA). The positive rate of surface antigen was calculated by FlowJo (LLC, Ashland, OR, USA) in units of percent.
4
Cell Cycle Analysis and Neutrophil Differentiation
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Cell cycle profiles were analyzed by propidium iodide staining as described in detail elsewhere18 (link). The number of apoptotic cells in the population was estimated by the Annexin-V Alexa488/PI kit (Invitrogen) according to the provider’s recommendations. Maturation status of the differentiating neutrophilic cell population was analyzed by immuno-detection of cell surface markers. The following combination of antibodies were used: PE mouse anti-human CD16b, APC mouse anti-human CD34, APC-Cy7 mouse anti-human CD11b, PE-Cy7 mouse anti-human CD14 (all from BD Biosciences) and FITC-anti-Annexin V (Invitrogen). Neutrophilic differentiation stages were identified by gating on subpopulations according to the expression of surface markers (Supplementary Fig. S6 )57 (link): CD34+ cells (CD34+, CD11b−, CD16b−), promyelocytes (CD34−, CD11b−, CD16b−), myelocytes (CD34−, CD11b+, CD16b−) and metamyelocytes (CD36−, CD11b+, CD16b+). Monocytes were identified according to the expression of CD14+. All samples were measured with a FACS cytometer (BD Biosciences) and analyzed by the FlowJo software. Data were statistically analyzed by χ2 test for each replicate as well as pairwise comparison by Student’s t-test in GraphPad Prism. Full methodical details are given in the Supplementary information online.
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