1000 sort-purified LT-HSCs from M2/HSB/Tn mice were cultured together with 10,000 MS-5 stromal cells in round-bottom 96-well plates together with SCF (100 ng/ml), TPO (100 ng/ml), Flt3L (50 ng/ml), IL7 (20 ng/ml), IL3 (10 ng/ml), IL11 (50 ng/ml), and GM-CSF (20 ng/ml) in αMEM with 1% Penicillin/Streptomycin and 10% FCS (Thermo Fisher) for two weeks, changing the media 24h after sort and then every 48h (Becton Dickinson). Myeloid and lymphoid HSC progeny was FACS sorted after labelling with Gr-1, Mac-1, CD19 and B220 antibodies (eBiosciences). All growth factors and cytokines were mouse recombinant and purchased from Peprotech.
Mac 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mac-1 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for use in various scientific research and analysis applications. The core function of the Mac-1 is to perform specific tasks required in a laboratory setting, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ter119, by Thermo Fisher Scientific (13 mentions)
Facsaria 2, by BD (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Sca 1, by Thermo Fisher Scientific (3 mentions)
B220 antibodies, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Cd201, by Thermo Fisher Scientific (2 mentions)
Calibur, by BD (2 mentions)
Vybrant multicolor cell labeling kit, by Thermo Fisher Scientific (2 mentions)
Thy 1, by Thermo Fisher Scientific (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Facsvantage se, by BD (1 mentions)
C kit 2b8, by BD (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd117 microbeads, by Miltenyi Biotec (1 mentions)
Cd150, by Thermo Fisher Scientific (1 mentions)
Automacs machine, by Miltenyi Biotec (1 mentions)
Summit software, by Agilent Technologies (1 mentions)
Streptavidin apc cy7, by BioLegend (1 mentions)
Il 7r, by Thermo Fisher Scientific (1 mentions)
22 protocols using mac 1
1
Expansion of Murine Hematopoietic Stem Cells
2
Immunophenotypic Analysis of Murine Hematopoietic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow (BM) and spleen cells were stained with either PE- or APC-conjugated monoclonal antibodies specific for Ter119, CD71, CD61, CD41, Mac-1, Gr-1, B220 or Thy-1 (purchased from eBioscience or Biolegend, San Diego, CA, USA) for 20 min on ice. Flow cytometry was performed with an LSRII (Beckton-Deckinson, San Diego, CA, USA) and analyzed by using FlowJo software (TreeStar, Ashland, OR, USA). For stem/progenitor analysis, BM or spleen cells were stained with antibodies against c-Kit, Sca-1, CD34, CD16/32 (FcγR II/III), and antibodies against lineage (Lin) markers including CD3, CD4, CD8α, CD19, B220, Gr-1, CD127 and Ter119 (from eBioscience or Biolegend, San Diego, CA, USA) for 1 hour on ice followed by flow cytometric analysis.
3
Characterization of Murine Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the expression of TLT-6 in primary cells, cells were harvested from the bone marrow and bone marrow-derived and peritoneal macrophages were collected, and washed twice in staining buffer (PBS, 0.01% NaN3, and 2% FBS). Cells were stained with fluorescence (FITC, PE, or APC)-, biotin-conjugated hematopoietic-specific monoclonal antibodies (CD4, CD3, CD8, B220, Gr-1, Mac-1, c-Kit, Sca-1, CD11b, Ter-119, and CD11c), or matched isotype controls purchased from eBiosciences (Sand Diego, CA, USA). Macrophages derived from the peritoneum or bone marrow were activated by LPS. Red cells were lysed, and nonspecific binding was blocked by preincubation with nonconjugated antibody to CD16/32. Dead cells were excluded by propidium iodide staining. Whole cells stained were analyzed using FACSVantage SE (BD Biosciences).
4
Competitive Homing Assay for Hematopoietic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse competitive homing experiments were performed as described32 (link). In brief, BM cells from Fancb−/y or their male WT littermates (CD45.2+) were labeled with DiO dye at a density of 2 × 106 cells per ml at 37 °C for 30 min. In parallel, BM cells from BoyJ mice (CD45.1+) were labelled with DiD dye (1:200) at 37 °C for 30 min. DiO and DiD were purchased from Vybrant Multicolor Cell-Labelling Kit (Molecular Probes, V-22889). After labelling, dyes were washed off. The DiO-labelled CD45.2 and DiD-labelled CD45.1 cells were mixed at a 1:1 ratio and transplanted into lethally irradiated (11 Gy one day before transplantation) CD45.1 recipients (2.5 × 106 from each donor). Sixteen hours after transplant, the recipients were euthanized and the BM was analyzed by flow cytometry for both DiO/DiD and surface lineage markers (Gr1, Mac1, B220, CD3, Ter119, from Ebioscience) and c-Kit (2B8, BD Biosciences). The ratio between the percentages of DiO+ (donor) and DiD+ (competitor) cells within different cell populations was quantified.
5
Murine Hematopoietic Stem Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow cells were harvested by crushing two tibias, two femurs, two pelvises, and one spine from each mouse. Bone marrow cells were enriched for immature cells using anti–mouse CD117 MicroBeads and an autoMACS machine (both Miltenyi Biotec) per manufacturer’s instructions. c-Kit–enriched populations were stained with antibodies against lineage markers (B220, CD3, Gr-1, Mac-1, and Ter119), c-Kit, Sca-1, CD150, CD16/32, and CD34 (eBioscience) as previously described (McGowan et al., 2011 (link)). Stained samples were either analyzed or sorted using a FACSAria II (BD). For all experiments in which c-Kithi or c-Kitlo HSCs were purified, they were double-sorted to ensure >95% purity. To calculate the frequency of hematopoietic precursors, two femurs and two tibias were flushed into 1x PBS containing 2.5% fetal calf serum (Hyclone). Bone marrow aspirations were performed on mice under isoflurane anesthesia per IACUC-approved protocol. Aspirates were treated with ACK lysis buffer and stained in PBS/2.5% fetal calf serum with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, Flk2, CD34, CD48, and CD41 for analysis on hematopoietic stem and progenitor cells. For myeloid progenitors, bone marrow cells were stained with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, CD41, CD105, and CD71, as described by Pronk et al. (2007) (link).
6
Purification of Mouse CD34-KSL HSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse CD34−KSL HSCs were purified from BM cells of 8-10-week-old mice. The cells were stained with an antibody cocktail consisting of biotinylated anti-Gr-1, −Mac-1, −CD4, −IL-7R, and -Ter-119 (eBioscience, San Diego, CA), and -B220 and -CD8 monoclonal antibodies (BioLegend, San Diego, CA) (lineage-marker cocktail). Lineage-positive cells were depleted with anti-Biotin MicroBeads (Miltenyi Biotec, Auburn, CA) and LS columns (Miltenyi Biotec). The remaining cells were further stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (BD Bioscience, California, CA), phycoerythrin (PE)-conjugated anti-Sca-1 (eBioscience), and allophycocyanin (APC)-conjugated anti-c-Kit antibodies (BioLegend). Biotinylated antibodies were detected with streptavidin-APC-Cy7 (BioLegend). Analysis and cell sorting were performed on a MoFlo using Summit software (Dako, Glostrup, Denmark) and results were analyzed with FlowJo software (Tree Star, Ashland, OR).
7
Isolation and Characterization of Murine Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophages and neutrophils were isolated from blood of Floxed and Fp38αKO mice. RBCs were lysed using ACK lysis buffer. A single-cell suspension was used for staining cell surface markers F4/80, Gr-1, and Mac-1 (eBioscience) following standard protocols, and data acquisition was performed using a FACSAria II cytometer (BD). Flow cytometric data were analyzed with FLOWJO software.
8
Isolation and Purification of Murine LSK Cells
9
Enrichment of Hematopoietic Progenitor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Progenitor cells were enriched by depleting lineage-positive cells using magnetic cell sorting. Whole FL cells were incubated with biotin-labeled monoclonal antibodies: B220, CD3, Gr-1, and Ter119, all these antibodies are from mouse hematopoietic lineage biotin panel (88-7774-75, ebioscience). Cells were then washed with MACS buffer (PBS, 2 mM EDTA, and 0.5% BSA), followed by staining with streptavidin-conjugated magnetic beads (558451, BD). After staining for 20 min at 4°C, the cells were washed and suspended in MACS buffer, and the lineage-positive cells were depleted by the magnetic system. The lineage-depleted progenitor cells were then incubated with APC-labeled lineage markers, including B220 (17-0452, eBioscience), CD3 (17-0032, eBioscience), Gr-1 (17-5931, eBioscience), Mac-1 (17-0112, eBioscience), PE-labeled anti-CD150 antibody (12-1502, eBioscience), and FITC-labeled anti-CD48 antibody (11-0481, eBioscience). After incubation for 30 min at 4°C, the cells were washed with PBS, suspended with IMDM, and supplemented with 1% BSA. Flow-sorting was performed on an LSR Fortessa flow cytometer (BD).
10
Evaluating DUX4/IGH Lymphoid Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A lineage depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to isolate mouse bone marrow lineage‐negative (Lin−) cells. The flow cytometry (FACS) was used to obtain Lin−/c‐Kit Low cells. The bone marrow cells were then infected with the retroviruses containing MigR1‐HA‐DUX4/IGH or mutants. The transfected cells were co‐cultured with an OP9 monolayer for 5 days in Iscove's Modified Dubecco's Medium (IMDM) containing 20% FBS, 50 ng/mL FMS‐like tyrosine kinase 3 ligand (Flt3L), 10 ng/mL Interleukin 3 (IL‐3), 10 ng/mL IL‐6, 50 ng/mL IL‐7, and 20 ng/mL recombinant human stem cell factor (SCF). The lymphoid lineage differentiations by DUX4/IGH and mutants were evaluated by FACS using antibodies against mouse CD19 (561737, BD Pharmingen, Franklin Lakes, NJ, USA) and Mac‐1 (48‐0112‐82, eBioscience, San Diego, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!