Mouse bone marrow cells were isolated from femurs and tibiae. Polymorphonuclear cells (PMN) were purified by negative selection (Stemcell Technologies) according to the manufacturer's directions. For inflammatory neutrophils, mice were injected intraperitoneally (i.p.) with 1 ml of 4% of thioglycolate, 24 h later peritoneal contents were collected and neutrophils were purified by positive selection using biotinylated anti-Ly-6G (Biolegend) and MACS streptavidin-microbeads (Miltenyi Biotec), following the manufacturer's instructions.
Biotinylated anti ly 6g
Manufactured by BioLegend
Biotinylated anti-Ly-6G is a lab equipment product used for immunological studies. It is a monoclonal antibody that binds to the Ly-6G antigen, which is expressed on the surface of granulocytes. The biotinylation of the antibody allows for detection and visualization through streptavidin-based methods.
Automatically generated - may contain errors
Lab products found in correlation
Macs streptavidin microbeads, by Miltenyi Biotec (2 mentions)
Negative selection, by STEMCELL (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Aria fusion cell sorter, by BD (1 mentions)
Alexa fluor 488 conjugated f4 80, by BioLegend (1 mentions)
Pe conjugated anti 1 a 1 e, by BioLegend (1 mentions)
Percp cy5.5 conjugated anti cd11c, by BioLegend (1 mentions)
Pe cy7 conjugated anti cd86, by BioLegend (1 mentions)
Pe conjugated anti siglec f, by BD (1 mentions)
Alexa fluor 488, by BioLegend (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
Dynabeads untouched mouse t cells kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin microbeads, by BD (1 mentions)
Pe selection cocktail, by STEMCELL (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
6 protocols using biotinylated anti ly 6g
1
Isolation and Purification of Mouse Neutrophils
2
Neutrophil and B-Cell Purification
3
Isolation of Mouse Bone Marrow PMN
4
Isolation and Functional Assays for Myeloid and NK Cells
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For quantitative real-time PCR, immunoblotting, and in vitro functional assays of myeloid cells (i.e., differentiation, survival, or chemokine secretion), neutrophils were purified from BM of C57BL/6J or CIS−/− mice by stepwise immunolabeling with biotinylated anti-Ly6G (Biolegend), anti-biotin microbeads, and positive selection with LS columns (Miltenyi Biotec). Subsequently, BM monocytes were isolated by stepwise immunolabeling with biotinylated anti-CD115 (Biolegend), anti-biotin microbeads, and positive selection with LS columns (Miltenyi Biotec). Cells of at least 95% purity were used for stimulation with recombinant G-CSF or recombinant GM-CSF (100 ng/ml) for indicated times.
NK cells from mouse spleen homogenates were enriched with an NK cell isolation kit (Miltenyi Biotec) and sorted (PI– CD3– CD19– TCRβ– F4/80– NK1.1+ NKp46+ CD49b+ CD49a–) on an ARIA Fusion cell sorter (BD Biosciences) to achieve a final purity of 99–100%. NK cells were cultured at 37°C in RPMI 1640 medium containing 10% FCS for 48 h. To assess cytokine production, NK cells were stimulated with 10 ng/ml of rIL-15, 50 ng/ml of rIL-18, and 50 pg/ml of rIL-12. At the endpoint, supernatant was recovered and IFN-γ and GM-CSF levels were measured by ELISA.
NK cells from mouse spleen homogenates were enriched with an NK cell isolation kit (Miltenyi Biotec) and sorted (PI– CD3– CD19– TCRβ– F4/80– NK1.1+ NKp46+ CD49b+ CD49a–) on an ARIA Fusion cell sorter (BD Biosciences) to achieve a final purity of 99–100%. NK cells were cultured at 37°C in RPMI 1640 medium containing 10% FCS for 48 h. To assess cytokine production, NK cells were stimulated with 10 ng/ml of rIL-15, 50 ng/ml of rIL-18, and 50 pg/ml of rIL-12. At the endpoint, supernatant was recovered and IFN-γ and GM-CSF levels were measured by ELISA.
5
Isolation and Characterization of Immune Cells
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Iliac lymph nodes were excised from immunized mice and immediately immersed in Hank's Balanced Salt Solution medium and put on ice. Lymph nodes were then treated with 1 mg/mL Collagenase D (Roche Diagnostics, Indianapolis, IN) at 37 °C for 20 min to release cells. Cells were washed with PBS containing 2 mM EDTA, blocked with 1% normal rabbit serum, and stained with the following antibodies and reagents: Zombie Violet, biotinylated anti-Ly6G, biotinylated anti-Ly6C, Alexa Fluor 488 or PerCP-Cy5.5-conjugated anti-CD11c, PE-conjugated anti-I-A/I-E, PE/Cy7-conjugated anti-CD86, Alexa Fluor 488-conjugated F4/80 (all from BioLegend, San Diego, CA), and PE-conjugated anti-Siglec F (BD Bioscience, San Jose, CA). Biotinylated antibodies were detected with Alexa Fluor 488 or APC-conjugated streptavidin. After staining, cells were fixed in 2% paraformaldehyde. Cells were examined in a Canto II flow cytometer (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (FlowJo, Eugene, OR).
6
Isolation and Characterization of Murine Myeloid Cells
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Bone marrow cells were isolated from the tibia and femur and cultured in RPMI1640 medium with 10% FBS, 1% penicillin‐streptomycin, 55 µm β‐mercaptoethanol and 10% L929 conditioned media containing macrophage‐colony stimulating factor (M‐CSF) for 6 days to harvest BMDMs. Mouse T cells were isolated from spleens by using the Dynabeads Untouched Mouse T Cells Kit (Thermo Fisher). CD11b+Ly6ChiLy6G– cells were sorted on a MoFlo Astrios instrument. For CD11b+Ly6G+ isolation, cells were labeled with biotinylated anti‐Ly6G (BioLegend), incubated with streptavidin microbeads (BD), and separated on magnetic columns (Stemcell). For specific cell isolation from splenocytes, pDCs were isolated using anti‐mPDCA‐1 microbeads from Miltenyi Biotec (Auburn, CA). After pDCs isolation, macrophages were isolated with CD11b microbeads from Miltenyi Biotec, cDCs were isolated with mouse CD11c PE labeling and followed by PE selection cocktail from STEMCELL technologies, following the manufacturer's protocol. RNAs from pDCs, cDCs, and macrophages were isolated using TRIzol reagent (Invitrogen) and subjected to semi‐quantitative PCR analysis of 18S rRNA by using specific primer.
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