Biotin mouse lineage panel

Manufactured by BD

Sourced in United States

The Biotin mouse lineage panel is a lab equipment product that allows for the identification and characterization of different cell types in mouse samples. It provides a way to detect the presence and abundance of various cell lineages.

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Biotin-conjugated mouse lineage panel Mouse Lineage Panel

8 protocols using biotin mouse lineage panel

Isolation and Sorting of Murine HSC Subsets

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Murine lineage negative (Lin⁻) cells were isolated from the bones of Ptprc (CD45.1) mice. The mice were sacrificed and the hind limbs (femur and tibia) were dissected out. The bone marrow was flushed into IMDM supplemented with 5% FBS, and the MNCs were isolated by density gradient centrifugation (HiSep™ Hi Media, Mumbai, India). For the isolation of Lin⁻ cells the MNCs were first incubated with biotin-labeled anti-mouse lineage antibody cocktail (Additional file 1: Table S1) prepared from biotin mouse lineage panel (BD Pharmingen, New Jersey, USA) and then the antibody-bound lineage-positive cells were depleted using Dynabeads® biotin binder (Invitrogen, Carlsbad, California, USA).
For sorting of LSK-CD34⁻ and LSK-CD34⁺ cells, the MNCs were incubated with antibodies (Additional file 1: Table S1) against lineage markers, Sca-1, c-Kit, and CD34. Cells were washed to remove unbound antibodies and LSK-CD34⁻ and LSK-CD34⁺ cells were sorted on FACS ARIA II (Becton Dickinson, New Jersey, USA).

Jalnapurkar S., Singh S., Devi M.R., Limaye L, & Kale V. (2016). Nitric oxide has contrasting age-dependent effects on the functionality of murine hematopoietic stem cells. Stem Cell Research & Therapy, 7, 171.

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Murine Hematopoietic Stem Cell Immunophenotyping

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Spleen and bone marrow were harvested from littermates, processed into single cell suspensions, and immunophenotyping for mature lymphoid, myeloid and HSPC cell lineages was performed as previously described (25 (link),26 (link)). The following antibodies were used: biotin mouse lineage panel (BD; includes CD11b, Gr1, CD3, B220, Ter119), biotin-CD4 (RM4–5; eBioscience), biotin-CD8a (53–6.7; eBioscience), biotin-CD19 (eBio1D3; eBioscience), pacific blue-conjugated streptavidin (Invitrogen), BUV395 or FITC Sca1 (D7; BD), APC cKit (ACK2; eBioscience), PE Flt3 (A2F10; BioLegend), PECy7 CD150 (TC150–12F12.2; BioLegend), APC-Cy7 CD48 (HM48–1, BD). Samples were run on a Fortessa or Celesta instrument (BD Biosciences) and analyzed using FlowJo software. HSPC populations were defined as follows: lineage negative (CD19−, B220−, CD4−, CD8−, CD3−, Gr1−, CD11b−, Ter119−), LSK (lineage−, cKit+, Sca1+), MPP (LSK, Flt3+), ST-HSC (LSK, Flt3−, CD48−, CD150−), and LT-HSC (LSK, Flt3−, CD48−, CD150+).

Puccetti M.V., Adams C.M., Dan T.D., Palagani A., Simone B.A., DeAngelis T., Eischen C.M, & Simone N.L. (2019). miR-21 is required for hematopoietic cell viability following radiation exposure. International journal of radiation oncology, biology, physics, 104(5), 1165-1174.

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Immunophenotyping of Bone Marrow Progenitors

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Whole bone marrow was isolated and stained on ice with various antibody cocktails to identify each progenitor compartment. PE-CD11b and FITC-Ly6G (BD Science, San Jose, CA, USA) were used to detect the neutrophil populations, while PE-CD184 (BD Science, San Jose, CA, USA) was used to detect the CXCR4⁺ cell populations. Biotin-CD4, Biotin-CD8a, Biotin Mouse Lineage Panel, PE-CD135, V450-CD127, APC-CD117, PE-Cy7 Streptavidin (BD Science, San Jose, CA, USA), PE-Cy5.5 Sca1 (Abcam, Cambridge, UK), and AF700-CD16/32 (eBioscience, San Diego, CA, USA) were used to detect the HSCs and their differentiated cell populations. All the labeled cells were evaluated using an Aria II flow cytometer (BD Science, San Jose, CA, USA) or FC500 flow cytometer (Beckman, Brea, CA, USA), and the data were processed using FlowJo software (BD Science, San Jose, CA, USA). For detailed procedure, see Supplementary Figure 2.

Lei X., Zhi C., Huang W., Sun X., Gao W., Yin X., Zhang X., Liang C., Zhang H, & Sun F. (2020). Recombinant Ganoderma lucidum Immunomodulatory Protein Improves the Treatment for Chemotherapy-Induced Neutropenia. Frontiers in Pharmacology, 11, 956.

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Isolation and Analysis of Mouse LSK Cells

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Monocytes were isolated from total BM cells by Ficoll-Paque-PLUS (Cytiva, Tokyo, Japan) before dead cells were removed with a LIVE/DEAD Fixable Blue DEAD cell stain kit (Thermo Fisher Scientific Inc.). The samples were stained with a Biotin Mouse lineage panel (BD Biosciences, San Jose, CA, USA), followed by PECy7-conjugated streptavidin, APC-conjugated c-kit, and APCCy7-conjugated Sca-1 (all from BD Biosciences), and then LSK cells were isolated by a FACS Fusion (BD Biosciences). An RNeasy Plus Micro kit (Qiagen) was used to extract RNA from LSK cells, and then the samples were sent to Takara Bio Inc. (Shiga, Japan), where the RNA was amplified and microarray analysis was performed.

Kojima H., Katagi M., Okano J., Nakae Y., Ohashi N., Fujino K., Miyazawa I, & Nakagawa T. (2023). Complete remission of diabetes with a transient HDAC inhibitor and insulin in streptozotocin mice. Communications Biology, 6, 637.

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Mouse Bone Marrow Cell Isolation and Staining

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Mouse BM cells were obtained from femurs and tibias. Red blood cells were removed by incubation with Qiagen Buffer EL (cat# 79217) according to manufacturer’s protocol. Cells were resuspended in PBS with 0.5% BSA at a density of approximately 10⁷ cells/mL before proceeding to staining. For lineage staining, BD Pharmingen Biotin Mouse Lineage Panel (cat# 559971, RRID:AB_10053179) was used according to manufacturer’s instructions and followed by staining with 0.5 μl streptavidin-Pacific Blue (Invitrogen, cat# S11222) per 10⁶ cells. Additional antibodies are listed in Supplemental Table S3. Data were analyzed using FlowJo (Becton Dickinson and Company, Ashland, OR, USA; RRID:SCR_008520) and GraphPad Prism (GraphPad Software, La Jolla, CA, USA; RRID:SCR_002798).

Johnston G., Ramsey H., Liu Q., Wang J., Stengel K.R., Sampathi S., Acharya P., Arrate M., Stubbs M.C., Burn T., Savona M.R, & Hiebert S.W. (2020). Nascent transcript and single-cell RNA-seq analysis defines the mechanism of action of the LSD1 inhibitor INCB059872 in myeloid leukemia. Gene, 752, 144758.

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Isolation and Sorting of Murine LSKs

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BM cells were flushed from tibias and femurs of both legs of each donor into cold DMEM containing 2% FBS and 100ng/mL penicillin/streptomycin. Lineage-positive cells were depleted using the Lineage Cell Depletion Kit (Miltenyi Biotec, Germany). Lineage-negative cells were then stained with Biotin Mouse Lineage Panel (BD Pharmingen, USA), Streptavidin APC-Cy7 (BD Pharmingen, USA), Sca1-PE (BD Pharmingen, USA), and c-Kit-APC (Biolegend, USA) antibody mixture. LSKs were sorted by BD FACSAria (BD Biosciences, USA).

Chen Z., Xie Y., Liu D., Liu P., Li F., Zhang Z., Zhang M., Wang X., Zhang Y., Sun X, & Huang Q. (2021). Downregulation of miR-142a Contributes to the Enhanced Anti-Apoptotic Ability of Murine Chronic Myelogenous Leukemia Cells. Frontiers in Oncology, 11, 718731.

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Isolation and Culture of Hematopoietic Cells

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For isolation of bone marrow cells, bone marrow from femur and tibia bones was flushed into ice-cold PBS buffer. After red blood cells were lysed with ammonium chloride (STEMCELL 07850), bone marrow cells were resuspended with PBS and filtered into a round bottom tube with cell strainer cap. The hematopoietic cell lineages were stained with biotin mouse lineage panel (BD Biosciences 559971) then isolated by magnetic beads separation (Zhang et al., 2013 (link)). For c-Kit positive cells, mouse hematopoietic cells were isolated from bone marrow using MACS CD117 isolation system. Purified bone marrow cells were cultured in RPMI medium supplemented with 10% FBS, L-glutamine, penicillin streptomycin, recombinant mouse SCF, IL-3, and IL-6. Control or Pdxk targeting shRNAs were delivered by lentivirus. Spin infections were performed at 37°C and 1500 rpm for 60 min. After spin infection, viral supernatants were substituted with leukemic cell culture medium. For compound treatment, isoniazid or 4'-O-methylpyridoxine was added in culture medium at indicated concentration.

Chen C.C., Li B., Millman S.E., Chen C., Li X., Morris JP I.V., Mayle A., Ho Y.J., Loizou E., Liu H., Qin W., Shah H., Violante S., Cross J.R., Lowe S.W, & Zhang L. (2020). Vitamin B6 addiction in acute myeloid leukemia. Cancer cell, 37(1), 71-84.e7.

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T Cell Activation and B Cell Co-culture

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0.2x10⁵ magnetic‐activated cell sorting (MACS)‐purified (Pan TC Isolation Kit, Miltenyi, Germany) carboxyfluorescein succinimidyl ester (CFSE)‐stained (BioLegend) T cells from naïve C57BL/6 mice treated with DMF or control were plated in 96‐well plates and stimulated with 0, 0.125 or 0.25 µg/mL LEAF™ purified anti‐mouse CD3 (BioLegend) and 0.5 µg/mL LEAF™ purified anti‐mouse CD28 antibodies (BioLegend) for 72 h. 0.5 × 10⁶ MACS‐purified B cells (Biotin mouse lineage panel, BD Biosciences, Franklin Lakes, NJ, USA) from spleens of DMF‐ or control‐treated immunized C57BL/6 mice were plated in 96‐well plates and co‐cultured with 0.2 × 10⁵ MACS‐purified CFSE‐stained 2D2 T cells, stimulated with MOG protein_1‐117 and evaluated 65 h thereafter.

Traub J., Traffehn S., Ochs J., Häusser‐Kinzel S., Stephan S., Scannevin R., Brück W., Metz I, & Weber M.S. (2019). Dimethyl fumarate impairs differentiated B cells and fosters central nervous system integrity in treatment of multiple sclerosis. Brain Pathology, 29(5), 640-657.

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