Hematopoietic stem cells and uncommitted progenitors (LKS +: Lin − Kit + Sca-1 +) and committed progenitors (LKS −: Lin − Kit + Sca-1 − CD16/32 +/− CD34 +/− representing CMP: common myeloid progenitors, GMP: granulocytic-macrophage progenitors and MEP: myelo-erythroid progenitors) were isolated by staining with a biotinylated cocktail to label and eliminate mature cells (anti-CD5, anti-CD11b, anti-B220, anti-7-4, anti-Gr1, anti-Ter119, Miltenyi Biotec), followed by eFluor 450-streptavidin, APC-anti-c-Kit, PE-anti-Sca-1, PE-Cy7-anti-CD16/32 and Alexa-Fluor 700-anti-CD34 to label HSPC. LKS + sub-populations were labeled using biotin-conjugated anti-mouse lineage antibodies (Miltenyi Biotec) followed by PE-Texas Red-streptavidin (eBioscience), APC-anti-c-Kit, PECy7-anti-Sca-1, FITC-anti-CD34 and PE-anti-Flk2 (LT-HSC: LKS + CD34 − Flk2 −, ST-HSC: LKS + CD34 + Flk2 − and MPP: LKS + CD34 + Flk2 +). Then cell sorting was performed on a BD Influx cell sorter.
Anti ter 119
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-Ter-119 is a monoclonal antibody that binds to the Ter-119 antigen, which is expressed on the surface of erythroid cells. The Ter-119 antigen is a glycophorin-associated protein that is widely used as a specific marker for the erythroid lineage in flow cytometric analysis and cell sorting applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b, by Miltenyi Biotec (6 mentions)
Anti gr1, by Miltenyi Biotec (4 mentions)
Anti cd3, by Miltenyi Biotec (4 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (4 mentions)
Anti cd45r, by Miltenyi Biotec (4 mentions)
Il 33, by Thermo Fisher Scientific (3 mentions)
Anti cd21 cd35, by Miltenyi Biotec (3 mentions)
Anti cd49, by Miltenyi Biotec (3 mentions)
Anti cd5, by Miltenyi Biotec (2 mentions)
Anti cd11c, by Miltenyi Biotec (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Anti cd3 and anti cd28, by BioLegend (2 mentions)
Influx cell sorter, by BD (1 mentions)
Biotin conjugated anti mouse lineage antibodies, by Miltenyi Biotec (1 mentions)
Anti 7 4, by Miltenyi Biotec (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti ter 119
1
Isolation and Characterization of Murine Hematopoietic Stem Cells
2
Murine Hematopoietic Cell Analysis
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TRI reagent was from Sigma-Aldrich (Madrid, Spain). Antibodies for the detection of murine CD135 (Flt3), CD34, CD16/32, and CD127 (IL-7Rα) were from eBioscience (Barcelona, Spain). The Lineage Cell Depletion Kit for mice, FcR Blocking reagent and murine flow cytometry antibodies (CD3e, CD4, CD8a, CD11b, CD19, CD43, CD45R (B220), CD117 (c-Kit), anti-Gr1, anti-Sca-1, anti-Ter-119, anti-IgM) were from Miltenyi Biotec (Madrid, Spain). Super Script II reverse transcriptase and RNase OUT ribonuclease inhibitor were from Invitrogen and Thermo Fisher Scientific (Madrid, Spain). GoTaqR qPCR master mix was from Promega (Madrid, Spain).
3
Isolation of Murine B Cell Subsets
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Human PBMC purified B cells were isolated by Ficoll-Paque, followed by anti-CD19 magnetic beads (Miltenyi Biotec). Splenic naive B cells were purified using a B cell isolation kit by negative selection (Miltenyi Biotec). Splenic Pre-GC and GC B cells were first enriched by streptavidin magnetic beads conjugated with a mixture of biotinylated anti-CD11b, anti-CD11c, anti-IgD, anti-CD138, anti-CD3, and anti-Ter119 (Miltenyi Biotec), followed by cell sorting using a BD FACSAria III Cell Sorter. Splenic Pre-GC cells defined as B220+ IgD− CD38+ GL7+ cells were isolated from PyNL-infected mice on day 9 after infection. Splenic mature GC B cells defined as B220+ IgD− CD38− GL7+ cells were purified from mice recovered from PyNL infection after day 28 infection.
4
Isolation of Purified Platelets
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Platelets were isolated as recently described in [23 (link)]. To remove residual red blood cells and leukocytes for pure platelet preparations, cells were incubated with anti-Ter-119 and anti-CD45 beads (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively.
5
ILC2 Isolation and Viability Assay
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ILC2s were sorted by magnetic beads combined with flow cytometry from mice spleen. Mice splenic cell suspension was stained by Biotinbinding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, antiCD3, anti-CD11b, and anti-CD49) (Miltenyi Biotec, Belgish-Gladbach, Germany), and then combined with Streptavidin-MicroBeads, the negative cells were enriched by magnetic column. This step was mainly to remove most of the lineage-expressing cells in spleen, thus enriching lineage-negative cells. The enriched cells were stained with FITC-lineage (eBioscience, San Diego, CA, USA), PE-CD90.2, and APC-ST2 (R&D Systems, Inc., Minneapolis, MN, USA), then further sorted by flow cytometry. The sorted cells (1 × 105 cells per well in a 96-well plate) were cultured in 1640 medium supplemented with 10% FBS, 10 ng/mL IL-7, 10 ng/mL IL-2, and10 ng/mL IL-33 (Peprotech, Rocky Hill, USA), 10 U/mL Penicillin and 10 U/mL streptomycin. Three days later, ILC2s were cultured with IgG-NPs at 0, 2, 4, 8 and 16μg/mL for 24h and 48 h. The cell viability was determined using cell counting kit (CCK8) (Dojindo, Japan) according to the manufacturer’s instructions. The plates were scanned at 450 nm for absorbance using a spectrophotometer (BioTek, Winooski, VT, USA). Each data point was measured for the average from six duplicates. The experiments were repeated independently for 3 times.
6
Multiparametric Flow Cytometry Analysis of NK Cells
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Single cell suspensions of splenocytes or peripheral blood were prepared using the standard techniques. Fc receptors were blocked by using 2.4G2 mAb prior to surface staining with the indicated antibodies. The negative selection of NK cells was determined by staining with cell surface monoclonal antibodies (mAbs), including anti-CD19, anti-CD4, anti-CD8a, anti-CD5, anti-Gr1, and anti-Ter-119 (Miltenyi Biotech). The surface phenotype of NK cells was determined by staining with mAbs, including anti-CD3-PE and anti-NK1.1-APC (Biolegend, San Diego, CA). Intracellular IFNγ was measured by pacific blue-conjugated anti-IFNγ (Biolegend) as described previously [47 (link)]. Dead cells were excluded by propidium iodine (PI) staining and live cells were gated on PI-negative cells. The NK cells of donors or recipients were distinguished by using anti-CD45.1 mAb (A20, BD Biosciences, San Jose, CA), FITC-34-2-12 (H-2Dd mAb, BD Biosciences), or Bio-KH95 (anti-H-2Db mAb, BD Biosciences). Data acquisition was performed with FACS Calibur flow cytometer (BD Pharmingen, San Jose, CA) using a CellQuest software (BD Biosciences). Data analysis was performed using a FlowJo software [48 (link)].
7
Isolation and Culture of Mouse ILC1s
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ILC1s were isolated from the spleens of C57BL/6 mice by combining magnetic beads and flow sorting. Mouse spleen cells were used to generated single-cell splenocyte suspensions[24 ]. The obtained cells were incubated with biotin-conjugated antibodies (anti-CD3ε, anti-CD45R, anti-Gr-1, anti-CD11c, anti-CD11b, anti-Ter119, anti-TCR-αβ, and anti-FCεRI; Miltenyi, Belgish, Germany) to enrich lineage-negative cells following the Miltenyi magnetic bead cell isolation protocol. Enriched lineage-negative single cell suspensions were stained with an anti-mouse lineage cocktail (BioLegend, 145-2C11, RB6-8C5, RA3-6B2, Ter-119, and M1/70), anti-mouse CD127 (BioLegend, A7R34), anti-mouse NK1.1 (BioLegend, PK136), and anti-mouse NKp46 (BioLegend, 29A1.4) for flow cytometry sorting (BD FACS Melody) of lineage− CD127+ NK1.1+ NKp46+ cells (which were considered ILC1s); these cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 0.5 ng/ml IL-7 (Gibco, PMC0071).
8
Isolation and Culture of Murine CD8+ T Cells and ILC2s
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Murine CD8 + T cells were isolated from tumor tissue using the tumor in ltration CD8 + T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was con rmed by ow cytometry (96%). Sorted CD8 + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and ow cytometry. Brie y, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by ow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
ILC2s were sorted from murine spleen by a combination of microbeads and ow cytometry. Brie y, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by ow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
9
Isolation and Expansion of Murine CD8+ T Cells and ILC2s
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Murine CD8 + T cells were isolated from tumor tissue using the tumor infiltration CD8 + T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was confirmed by flow cytometry (96%). Sorted CD8 + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
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