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Cd11b microbead kit

Manufactured by Miltenyi Biotec
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The CD11b MicroBead kit is a laboratory product designed for the isolation and enrichment of CD11b-positive cells from various sample sources, such as peripheral blood, bone marrow, or tissue. The kit utilizes magnetic beads coated with antibodies specific to the CD11b surface marker to facilitate the separation and purification of the target cell population.

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Lab products found in correlation

Ls column, by Miltenyi Biotec (5 mentions) Alexafluor 647 α cd102 3c4 mic2 4, by BioLegend (4 mentions) Histopaque 1077 and 1119, by Merck Group (2 mentions) Anti ly6g microbead kit, by Miltenyi Biotec (2 mentions) αcd16 32, by BioLegend (2 mentions) Liveblazer fret b g loading kit with ccf4 am, by Thermo Fisher Scientific (2 mentions) Apc cy7 α cd19 1d3, by BD (2 mentions) Redfluor 710 α cd11b m1 70, by BioLegend (2 mentions) Apc cy7 α cd11b m1 70, by BioLegend (2 mentions) R718 α cd102 3c4 mic2 4, by BD (2 mentions) Novocyte 3000, by Agilent Technologies (2 mentions) Neural tissue dissociation kit, by Miltenyi Biotec (2 mentions) Facsaria 2 sorp, by BD (2 mentions) Fc block α cd16 32, by BioLegend (2 mentions) Pe cy7 α cd19 1d3, by BD (2 mentions) Fitc α cd11b m1 70, by BD (2 mentions) Bv510 α f4 80 bm8, by BioLegend (2 mentions) Bv711 α siglec f e50 2440, by BD (2 mentions) Violetfluor 450 α ly 6g gr1 rb6 8b5, by Cytek Biosciences (2 mentions) Cd3ε microbead kit, by Miltenyi Biotec (1 mentions)

11 protocols using cd11b microbead kit

1

CD3+ T and CD11b+ Cell Isolation

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CD3+ T cells were isolated from spleens by CD3ε MicroBead Kit and CD11b+ cells were isolated from BM by using CD11b MicroBead Kit (Miltenyi). Cells were positively selected by MACS technology according to manufacture’s protocol. Purity of all isolated cells ranged between 85–99%.
Leukocytes of different organs were isolated according to following protocols:
Kustermann M., Klingspor M., Huber-Lang M., Debatin K.M, & Strauss G. (2019). Immunostimulatory functions of adoptively transferred MDSCs in experimental blunt chest trauma. Scientific Reports, 9, 7992.
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2

Cardiac Fibroblast Interaction with IL-17Ra-Deficient Splenic Cells in EAM

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Spleens from WT or IL-17Ra–/– BALB/c mice on day 14 of EAM were dissected and mechanically disrupted. Cells were filtered through 40 mm cell strainers and washed. Histopaque 1119 and 1077 (Sigma) were used to isolate mononuclear cells. Anti-Ly6G MicroBead kit (Miltenyi Biotech) was used to deplete Ly6G+ cells, and CD11b MicroBead kit (Miltenyi Biotech) was used to positively enrich CD11b+ cells. Cells were sorted on an AriaIIu cell sorter. Sorted cells from IL-17Ra–/– BALB/c mice spleens were used for in vitro co-culture with primary cardiac fibroblasts, major outcomes were validated with sorted cells from WT BALB/c mice spleens co-cultured with cardiac fibroblasts. Sorted cells from WT BALB/c mice spleens were used in adoptive transfer experiments (see below).
Hou X., Chen G., Bracamonte-Baran W., Choi H.S., Diny N.L., Sung J., Hughes D., Won T., Wood M.K., Talor M.V., Hackam D.J., Klingel K., Davogustto G., Taegtmeyer H., Coppens I., Barin J.G, & Čiháková D. (2019). The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis. Cell reports, 28(1), 172-189.e7.
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3

Purification and Characterization of CD11b+ Cells

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PECs were collected and washed once. For latency experiments, cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). Total PECs were used for MHV68 acute infection experiments. CD11b+ cells or whole PECs were Fc blocked with α-CD16/32 (Biolegend) and stained with Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend), APC-Cy7-α-CD19 (1D3, BD Biosciences), redFluor 710-α-CD11b (M1/70, Tonbo) or APC-Cy7-α-CD11b (M1/70, Biolegend), and R718-α-CD102 (3C4 (MIC2/4), BD Biosciences). Cells were counted again and resuspended in 3 x 106 cells/mL before adding CC4F-AM (LiveBLAzer FRET-B/G Loading Kit with CCF4-AM, ThermoFisher Scientific). Cells were incubated for 1 hour, then washed with PBS and fixed with 2% formaldehyde. Samples were immediately run on a Novocyte 3000 (ACEA Biosciences).
Zarek C.M., Dende C., Coronado J., Pendse M., Dryden P., Hooper L.V, & Reese T.A. (2023). Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages. PLOS Pathogens, 19(10), e1011691.
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4

Isolation of Glioma-Associated Microglia/Macrophages

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Glioma associated brain microglia/macrophages were isolated from the human tumor resected tissues. Fresh tissue was dissociated immediately after resection using a neural tissue dissociation kit (MiltenyiBiotec, Bergisch Gladbach, Germany). Erythrocytes were lysed by adding 5 ml ammonium chloride solution. Thereafter, cells were resuspended in PBS containing 0.5% BSA and 2 mM EDTA. Magnetic sorting for CD11b+ cells was then performed by using CD11b MicroBead kit (MiltenyiBiotec) following the manufacturer´s instruction. Magnetic activated cell sorting (MACS) into CD11b negative (Flow-through) and CD11b positive (CD11b+) enriched cell populations was done using several MACS columns in series. Both CD11b negative and CD11b+ fractions were collected. A purity check was performed after MACS by flow cytometry analysis of a small fraction of the sorted populations.
Hu F., Ku M.C., Markovic D., Dzaye O.D., Lehnardt S., Synowitz M., Wolf S.A, & Kettenmann H. (2014). Glioma associated microglial MMP9 expression is up regulated by TLR2 signalling and sensitive to minocycline. International journal of cancer. Journal international du cancer, 135(11), 2569-2578.
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5

Astrocyte Purification via Negative Selection

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At the start of each experiment, astrocytes were further purified by magnetic microbead-based negative selection to remove microglia using the CD11b MicroBead kit (Miltenyi Biotec, Auburn, CA; Catalog # 130-093-634). After removing the medium, cells were briefly washed with cold Tris-EDTA (pH 7.4, Sigma Aldrich, Saint Louis, MO; Catalog # 93302) and then enzymatically detached from T75 flasks using cold 0.25% Trypsin-EDTA (Mediatech, Inc., Manassas, VA; Catalog # 25-053-CI). Next, the cells were pelleted by centrifugation (300 × g, 5 min) and resuspended in MACS buffer (0.5% w/v bovine serum albumin [BSA] in PBS). Cells were manually counted using a hemocytometer and then washed and incubated with CD11b MicroBeads according to the manufacturer’s protocol. Cell separation was performed using LS Columns (Miltenyi Biotec; Catalog # 130-042-401) and a MidiMACS Separator (Miltenyi Biotec; Catalog # 130-042-302) outfitted with 30-µm pre-separation filters (Miltenyi Biotec; Catalog # 130-041-407).
Rodgers K.R., Lin Y., Langan T.J., Iwakura Y, & Chou R.C. (2020). Innate Immune Functions of Astrocytes are Dependent Upon Tumor Necrosis Factor-Alpha. Scientific Reports, 10, 7047.
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6

Isolation and Characterization of Murine Myeloid Cells

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Three to four mice were pooled for each sample. PECs were collected and washed. Cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend). Samples were run on a BD FACS Aria II SORP and collected in D10 media (for LD-PCR). Afterward, the samples were counted and frozen for LD-PCR (in media).
Zarek C.M., Dende C., Coronado J., Pendse M., Dryden P., Hooper L.V, & Reese T.A. (2023). Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages. PLOS Pathogens, 19(10), e1011691.
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7

Cardiac Fibroblast Interaction with IL-17Ra-Deficient Splenic Cells in EAM

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Spleens from WT or IL-17Ra–/– BALB/c mice on day 14 of EAM were dissected and mechanically disrupted. Cells were filtered through 40 mm cell strainers and washed. Histopaque 1119 and 1077 (Sigma) were used to isolate mononuclear cells. Anti-Ly6G MicroBead kit (Miltenyi Biotech) was used to deplete Ly6G+ cells, and CD11b MicroBead kit (Miltenyi Biotech) was used to positively enrich CD11b+ cells. Cells were sorted on an AriaIIu cell sorter. Sorted cells from IL-17Ra–/– BALB/c mice spleens were used for in vitro co-culture with primary cardiac fibroblasts, major outcomes were validated with sorted cells from WT BALB/c mice spleens co-cultured with cardiac fibroblasts. Sorted cells from WT BALB/c mice spleens were used in adoptive transfer experiments (see below).
Hou X., Chen G., Bracamonte-Baran W., Choi H.S., Diny N.L., Sung J., Hughes D., Won T., Wood M.K., Talor M.V., Hackam D.J., Klingel K., Davogustto G., Taegtmeyer H., Coppens I., Barin J.G, & Čiháková D. (2019). The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis. Cell reports, 28(1), 172-189.e7.
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8

Isolation and Characterization of Murine Myeloid Cells

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Three to four mice were pooled for each sample. PECs were collected and washed. Cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend). Samples were run on a BD FACS Aria II SORP and collected in D10 media (for LD-PCR). Afterward, the samples were counted and frozen for LD-PCR (in media).
Zarek C.M., Dende C., Coronado J., Pendse M., Dryden P., Hooper L.V, & Reese T.A. (2023). Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages. PLOS Pathogens, 19(10), e1011691.
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9

Purification and Characterization of CD11b+ Cells

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PECs were collected and washed once. For latency experiments, cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). Total PECs were used for MHV68 acute infection experiments. CD11b+ cells or whole PECs were Fc blocked with α-CD16/32 (Biolegend) and stained with Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend), APC-Cy7-α-CD19 (1D3, BD Biosciences), redFluor 710-α-CD11b (M1/70, Tonbo) or APC-Cy7-α-CD11b (M1/70, Biolegend), and R718-α-CD102 (3C4 (MIC2/4), BD Biosciences). Cells were counted again and resuspended in 3 x 106 cells/mL before adding CC4F-AM (LiveBLAzer FRET-B/G Loading Kit with CCF4-AM, ThermoFisher Scientific). Cells were incubated for 1 hour, then washed with PBS and fixed with 2% formaldehyde. Samples were immediately run on a Novocyte 3000 (ACEA Biosciences).
Zarek C.M., Dende C., Coronado J., Pendse M., Dryden P., Hooper L.V, & Reese T.A. (2023). Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages. PLOS Pathogens, 19(10), e1011691.
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10

Isolation of Glioma-Associated Microglia/Macrophages

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Glioma associated brain microglia/macrophages were isolated from the human tumor resected tissues. Fresh tissue was dissociated immediately after resection using a neural tissue dissociation kit (MiltenyiBiotec, BergischGladbach, Germany). Erythrocytes were lysed by adding 5 mL ammonium chloride solution. Thereafter, cells were resuspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA. Magnetic sorting for CD11b+ cells was then performed by using CD11b MicroBead kit (MiltenyiBiotec) following the manufacturer´s instruction. MACS into CD11b negative (Flow-through) and CD11b positive (CD11b+) enriched cell populations was done using several MACS columns in series. Both CD11b negative and CD11b+ fractions were collected. A purity check was performed after MACS by flow cytometry analysis of a small fraction of the sorted populations (BD FACS Aria, BD Biosciences, San Jose, CA, USA).
Triller P., Bachorz J., Synowitz M., Kettenmann H, & Markovic D. (2020). O-Vanillin Attenuates the TLR2 Mediated Tumor-Promoting Phenotype of Microglia. International Journal of Molecular Sciences, 21(8), 2959.
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