Primary cells were isolated from 686LN xenografted tumors. After the dissection of tumors, single cells were isolated using tumor tissue dissociation kit (Miltenyi Biotech) then initially positively sorted using CD11b microbeads using a MACS seperator and MS columns (Miltenyi Biotech). CD11b+ population were further sorted using a FACS Aria cells were isolated using magnetic beads (Miltenyi Biotech). Macrophages were sorted using FACS Aria (BD Biosciences). Cells were labeled with fluorochrome-conjugated antibodies, PE/Cy7 anti-mouse Ly-6G, PerCP/Cy5.5 anti-mouse CD11b (Biolegend).
Percp cy5.5 anti mouse cd11b
Manufactured by BioLegend
PerCP/Cy5.5 anti-mouse CD11b is a fluorochrome-conjugated antibody that binds to the CD11b cell surface antigen expressed on mouse monocytes, macrophages, and granulocytes.
Automatically generated - may contain errors
Lab products found in correlation
Pe cy7 anti mouse ly 6g, by BioLegend (2 mentions)
Macs seperator, by Miltenyi Biotec (2 mentions)
Facs aria, by Miltenyi Biotec (2 mentions)
Ms column, by Miltenyi Biotec (2 mentions)
Magnetic bead, by Miltenyi Biotec (2 mentions)
Facsaria, by BD (2 mentions)
Tumor tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse cd45, by BioLegend (1 mentions)
Percp cy5.5 anti mouse cd45, by BioLegend (1 mentions)
Fitc anti mouse cd3ε, by BD (1 mentions)
Pe anti mouse cd69, by BioLegend (1 mentions)
Apc anti mouse cd11c, by BioLegend (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Trypsin edta solution, by Welgene (1 mentions)
Gfp sirna, by Bioneer (1 mentions)
Linear pei 25 kda, by Polysciences (1 mentions)
Dulbecco modified eagle medium (dmem), by Polysciences (1 mentions)
Apc cy7 anti mouse cd45, by BioLegend (1 mentions)
6 protocols using percp cy5.5 anti mouse cd11b
1
Isolation of Tumor-Associated Macrophages
2
Isolation and Sorting of Tumor-Associated Macrophages
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Primary cells were isolated from 686LN xenografted tumours. After the dissection of tumours, single cells were isolated using tumour tissue dissociation kit (Miltenyi Biotech) and then initially positively sorted using CD11b microbeads using a MACS seperator and MS columns (Miltenyi Biotech). CD11b+ population were further sorted using a FACS Aria cells were isolated using magnetic beads (Miltenyi Biotech). Macrophages were sorted using FACS Aria (BD Biosciences). Cells were labelled with fluorochrome-conjugated antibodies, PE/Cy7 anti-mouse Ly-6G and PerCP/Cy5.5 anti-mouse CD11b (Biolegend).
3
Multiparameter Immune Cell Analysis
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The following antibodies were used for NK cell analysis: FITC anti-mouse CD3ε (BD Biosciences, Cat# 553062, clone 145–2 C11), APC anti-mouse NK1.1 (eBioscience/Affymetrix, Cat# 17–5941, clone PK136), PerCp-Cy5.5 anti-mouse CD45 (BioLegend, Cat# 103132, clone 30-F11) and PE anti-mouse CD69 (BioLegend, Cat# 104507, clone H12F3). For pDCs analysis we used: PE-anti-mouse CD317 (PDCA-1) (BioLegend, Cat# 127104, clone 129C1), PerCP/Cy5.5 anti-mouse CD45 (BioLegend, Cat# 103132, clone 30-F11), APC anti-mouse CD11c (BioLegend, Cat# 117310, clone N418) and FITC anti-mouse/human CD45R/B220 (BioLegend, Cat# 103206, clone RA3-6B2). For myeloid cells we used: PerCp-Cy5.5 anti-mouse CD11b (BioLegend, Cat# 101228, clone M1/70), APC anti-mouse CD45 (BioLegend, Cat# 103112, clone 30-F11), FITC-anti-mouse Ly6C/Ly6G (Gr-1) (BioLegend, Cat# 108406, clone RB6-8 C5), PB anti-mouse F4/80 (BioLegend, Cat# 123123, clone BM8). For TLR7 expression analysis, we procured PE-anti-mouse TLR7 (BD Biosciences, Cat# 565557, clone A94B10). Exclusion of dead cells from the analysis was done based on staining with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific Cat# L34957), or DAPI (4ʹ6’-diamino-phenylindoledihydrochloride, ThermoFisher Scientific, Cat# D21490), or fixable viability dye eFluor780 (ThermoFisher Scientific, Cat# 65–0865-14).
4
Cytotoxicity Evaluation of Nanoparticles
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Citric
acid was purchased from Sigma-Aldrich.
Linear PEI (25 kDa) was purchased from Polysciences, Inc. DMEM (Dulbecco’s
modified Eagle’s medium), 0.25% trypsin–EDTA solution,
PBS (pH 7.4), FBS, and P/S were purchased from WelGENE Inc. CCK-8
(cell counting kit-8) was purchased from Dojindo Molecular Technologies,
Inc. CleanCap FLuc mRNA and CleanCap EGFP mRNA were purchased from
TriLink Biotechnologies. GFP siRNA was purchased from Bioneer. Recombinant
plasmid encoding RFP was purchased from Addgene. APC/Cy7 anti-mouse
CD45 and PerCP/Cy5.5 anti-mouse CD11b were purchased from Biolegend.
acid was purchased from Sigma-Aldrich.
Linear PEI (25 kDa) was purchased from Polysciences, Inc. DMEM (Dulbecco’s
modified Eagle’s medium), 0.25% trypsin–EDTA solution,
PBS (pH 7.4), FBS, and P/S were purchased from WelGENE Inc. CCK-8
(cell counting kit-8) was purchased from Dojindo Molecular Technologies,
Inc. CleanCap FLuc mRNA and CleanCap EGFP mRNA were purchased from
TriLink Biotechnologies. GFP siRNA was purchased from Bioneer. Recombinant
plasmid encoding RFP was purchased from Addgene. APC/Cy7 anti-mouse
CD45 and PerCP/Cy5.5 anti-mouse CD11b were purchased from Biolegend.
5
Isolation and Phenotyping of Tumor-Associated Macrophages
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The tumors were removed, cut into small pieces with a scalpel, added to HBSS culture containing 1 mg/mL collagenase (Sigma) and 0.1 mg/mL DNAase (Sigma), and incubated for 1 h at 37°C for digestion. The digests were sieved with 70 μM filters and the cells were collected by centrifugation and washed with PBS containing 2% FBS to prepare a tumor single‐cell suspension. Cell suspensions were then incubated with FcRblock (1 μg/test; BioLegend, #101319) for 10 min at 4°C, followed by treatment with CD45 (PE anti‐mouse CD45, 0.5 μg/test; BioLegend, #157603), F4/80 (Alexa Fluor® 488 anti‐mouse F4/80; 1 μg/test, BioLegend, #123119), CD11b (PerCP‐Cy5.5 anti‐mouse CD11b; 0.5 μg/test, BioLegend, #101227) antibodies at 4°C for 30 min staining. Then the cells were washed with PBS, fixed with Fixation Buffer (Fixation Buffer, BioLegend, #420801), and cell penetration was performed using 0.5% Triton X‐100. After cell resuspension with buffer containing 2% FBS in PBS, APC anti‐mouse CD206 antibody (0.5 μg/tests, BioLegend, #141707) was added and incubated for 30 min at room temperature and protected from light. Finally, the treated cell suspension was placed into a flow cytometer for detection.
6
Comprehensive Multiparameter Immune Profiling
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Alkaline Phosphatase (AP) anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG2c and IgG3 were acquired from Southern Biotech, while AP anti-mouse IgG was purchased from Bio-rad. BV421 anti-mouse I-Ab, PE anti-mouse I-Ad, BV421 anti-mouse H-2Kb, BV421 anti-mouse GL7, BV480 anti-mouse IgM, BV510 anti-mouse CD38, BB515 anti-mouse CD19, BV650 anti-mouse RORγt, PE CF594 anti-mouse GATA3, BB515 anti-mouse CD8α, and Alexa Fluor 647 (AF647) anti-mouse Bcl6 were obtained from BD Biosciences. FITC anti-mouse I-Ak, AF700 anti-mouse CD45, BV510 anti-mouse H-2Kd, PE anti-mouse H-2Kk, BV605 anti-mouse CD19, AF700 anti-mouse T cell receptor (TCR), AF700 anti-mouse CD11b, AF700 anti-mouse CD11c, BV650 anti-mouse IgD, BV421 anti-mouse CD3, BV510 anti-mouse CD4, BV605 anti-mouse Tbet, BV785 anti-mouse CD25, PerCP Cy5.5 anti-mouse CD19, PerCP Cy5.5 anti-mouse CD11c, PerCP Cy5.5 anti-mouse CD11b, and AF700 anti-mouse CD44 were purchased from Biolegend.
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