Cell sorting protocols were as previously described [28 (link), 32 (link)]. Briefly, bone marrow was flushed in PBS supplemented with 0.1% bovine serum albumin and 2 mM EDTA, filtered through 40 μm cell strainers, and subjected to red blood cell lysis in ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). The samples were stained with biotin anti-mouse Lineage Panel (BioLegend), PE-Cy7 cKit (clone 2B8, BioLegend), APC-Cy7 Sca-1 (D7, BioLegend), FITC CD48 (HM48-1, eBioscience), APC CD150 (TC15-12F12.2, BioLegend), PE Flt3 (A2F10, eBioscience), Brilliant Violet 421 CD34 (RAM34, BD Biosciences), and streptavidin-PECy5 (BioLegend). Cell sorting was performed on FACSAria, with the FACS Diva software (BD Biosciences).
Biotin anti mouse lineage panel
Manufactured by BioLegend
Sourced in United States
The Biotin anti-mouse lineage panel is a collection of antibodies that can be used to identify and distinguish different cell types within the mouse immune system. The panel includes antibodies that target specific cell surface markers, allowing for the detection and analysis of various lineages of immune cells, such as T cells, B cells, and myeloid cells. This tool is designed for use in flow cytometry applications to facilitate the study of mouse immune cell populations and their respective functions.
Automatically generated - may contain errors
Lab products found in correlation
Fitc cd48 hm48 1, by Thermo Fisher Scientific (2 mentions)
Streptavidin pecy5, by BioLegend (2 mentions)
Facsdiva software, by BD (2 mentions)
Streptavidin beads, by Miltenyi Biotec (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
β actin 8h10d10, by Cell Signaling Technology (1 mentions)
Apotracker green, by BioLegend (1 mentions)
Mouse il 4, by BioLegend (1 mentions)
Anti mouse cd3ε 145 2c11, by BioLegend (1 mentions)
Mojosort mouse cd8 t cell isolation kit, by BioLegend (1 mentions)
Mouse il 33, by BioLegend (1 mentions)
Mouse il 2, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
5 protocols using biotin anti mouse lineage panel
1
Isolation and Sorting of Hematopoietic Stem Cells
2
Lineage-depleted BM cell transplant
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BM cells from donor mice were collected and flushed with 1× PBS. Lineage depletion of the isolated BM cells was achieved by incubation with Biotin anti-mouse lineage panel (Biolegend) for 20 min, washed and incubated with streptavidin beads (Miltenyi) for 30 min, and then negatively isolated by MACS columns (Miltenyi) following the manufacturer’s instructions. Cell suspension containing ∼2 × 106 Lin− donor BM cells at a 1:1 ratio (WT: Vav1creBcl6fl/fl) were resuspended in 300 ml of 1xPBS, and then intravenously injected into lethally irradiated recipient mice. These mice were sacrificed and analyzed 8 wk after reconstitution.
3
Mouse Hematopoietic Stem Cell Isolation
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Mouse bone marrow was flushed from femurs, tibiae, humeri, and ilia of mice with a 26-G needle in PBS supplemented with 0.1% BSA and 2 mM EDTA, filtered through 40-μm cell strainers, and subjected to red blood cell lysis in ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA). The cells were stained with Biotin anti-mouse Lineage Panel (BioLegend) and lineage-positive cells depleted using biotin-binding Dynabeads (Life Technologies). The lineage-negative fraction was stained with PeCy7-cKit/CD117 (clone 2B8, BD Biosciences), APC-Sca1 (E13–161.7, BioLegend), FITC-CD48 (HM48-1, eBioscience), PE-CD150 (mShad150, eBioscience), and streptavidin-PECy5 (BioLegend). Propidium iodide or DAPI was added immediately before sorting for dead cell exclusion. Cell sorting was performed on the FACSAria instrument and was analyzed with FACS Diva software (BD Biosciences).
4
Isolation and Culture of Mouse T Cells
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Mouse IL-2 (catalog 575402), mouse IL-4 (catalog 547302), mouse IL-33 (catalog 580502), anti-mouse CD3ε (145-2C11), anti-mouse CD28 (37.51), human TGF-β1 (catalog 781802), anti-mouse IL-4 mAb (clone 11B11), anti-mouse IFN-γ mAb (XMG1.2), and Apotracker Green were purchased from BioLegend. Antibodies for TRAF4 (D1N3A), MyD88 (D80F5), p-mTOR (S2448, D9E), mTOR (7C10), p-AKT (S473, D9E), p-AKT (T308, D25E6), AKT (11E7), p-JNK (T183/Y185, 81E11), SAPK/JNK (no. 9252), p-p38 (T180/Y182, D3F9), p38 (D13E1), p-ERK1/2 (T202/Y204, D13.14.4E), Erk1/2 (137F5), p-IκBα (S32, 14D4), IκBα (L35A5), TRAF6 (D21G3), and β-Actin (8H10D10) were obtained from Cell Signaling Technology. Mouse T1/ST2 antibody (DJ8) and rabbit polyclonal against ST2 (ab228543) were obtained from MD Bioproducts and Abcam, respectively. AKT inhibitor VIII (CAS 612847-09-3) and LY3214996 (CAS 1951483-29-6) were purchased from Cayman Chemical. Primary mouse CD4+ T cells were cultured in complete TexMACS Medium (Miltenyi Biotec, 130-097-196) supplemented with 10% FBS, 50 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin. CD8+ cells and ILCs were isolated from spleen using MojoSort Mouse CD8 T Cell Isolation Kit and Biotin anti-mouse Lineage Panel from BioLegend.
5
Characterizing Mouse Immune Cell Subsets
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Flow cytometry was used to determine the cellular uptake of NPs and phenotypes of mouse immune cells using various combinations of the following fluorescence-labeled anti-mouse mAbs: CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), CD16/32(93), CD19 (6D5), CD25 (PC61), CD34(SA376A4), CD40 (3/23), CD45 (30-F11), CD80 (16-10A1), CD86 (GL-1), CD115(AFS98), CD117 (c-Kit, 2B8), F4/80 (BM8), I-A/I-E (M5/114.15.2), Ly-6C (HK1.4), NK1.1 (PK136), and Sca-1(D7). Biotin anti-mouse lineage panel from BioLegend (San Diego, CA, USA) was used in accordance with the manufacturer’s protocol. Single-cell suspensions were stained with flow cytometric antibodies for 30 min at 4°C. All samples were acquired on a BD LSR Fortessa (BD Bioscience, San Jose, CA, USA). The data were analyzed by FlowJo software (TreeStar, San Carlos, CA, USA).
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