Whole blood anticoagulated with heparin sodium (100 μL) was conjugated with fluoresceine isothiocyanate (FITC) rat anti-Mouse CD45 (553080, BD, USA), phycoerythrin (PE) rat anti-mouse Ly-6G and Ly-6C (553128), and Alexa Fluor 647 rat anti-mouse F4/80 (565853, BD) at room temperature for 30 min. Then, 2 mL of 1× red cell lysate was added for 10 min at room temperature in the dark and centrifuged for 5 min at 300× g. The supernatant was discarded, and 2 mL of phosphate-buffered saline (PBS) was added for resuspension and centrifuged at 300× g for 5 min. The supernatant was discarded again, and 500 μL of PBS was added, followed by analysis by flow cytometry (FCM; FACS Aria III, San Jose, CA, USA).
Fitc rat anti mouse cd45
Manufactured by BD
Sourced in United States
The FITC rat anti-mouse CD45 is a flow cytometry antibody that recognizes the CD45 antigen expressed on the surface of mouse hematopoietic cells. CD45 is a pan-leukocyte marker that plays a crucial role in the regulation of T and B cell antigen receptor signaling.
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Lab products found in correlation
Apc mouse anti human cd45, by BD (3 mentions)
Apc rat anti mouse ly 6g, by BD (2 mentions)
Pe rat anti mouse cd73, by BD (2 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Pe rat anti cd11b, by BD (1 mentions)
Dispase 2, by Merck Group (1 mentions)
Collagenase b, by Merck Group (1 mentions)
Ham s f10 media, by Wisent (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (1 mentions)
Pe rat anti mouse siglec f, by BD (1 mentions)
Pe cyanine7 anti mouse cd11c, by BioLegend (1 mentions)
Bv421 rat anti mouse f4 80, by BD (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Precision count beadstm, by BioLegend (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
Phospho b raf, by Cell Signaling Technology (1 mentions)
Gst tag, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
18 protocols using fitc rat anti mouse cd45
1
Immunophenotyping Murine Blood Leukocytes
2
Isolation and Identification of Lymphocytes and Macrophages
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Preparation of FACS tissue was carried out as previously described [62 ]. Cortex from perfused brains was mechanically homogenized and enzymatically digested before straining and separating through a Percoll gradient. Antibodies for CD3 (APC hamster anti-mouse CD3; 1:200; BD Bioscience #553,066), CD11b (PE rat anti-CD11b; 1:200; BD Bioscience #557,397), and CD45 (FITC rat anti-mouse CD45; 1:100; BD Bioscience #553,079) were added to cell suspensions and whole blood samples before the samples were washed and centrifuged, and the pellet resuspended in flow cytometry buffer. Samples were sorted on a FACS Aria™ Fusion cell sorter (Becton Dickenson), lymphocytes were identified as CD3 + , and macrophages were identified as being CD11b + CD45hi.
3
Flow Cytometric Characterization of Muscle Cell Populations
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The proximal third of the gastrocnemius was immediately processed for flow cytometry.22 Briefly, muscles were digested in Miltenyi gentleMACS C tube (Cat# 130‐093‐237, Miltenyi Biotec) containing Collagenase B (1.5 U/mL, Cat# 11088831001, Sigma‐Aldrich) and Dispase II (2 U/mL, Cat# 42613‐33‐2, Sigma‐Aldrich) in Ham's F10 media (Cat# 318‐050‐CL, Wisent Inc) by gentleMACS Octo Dissociator (Cat# 130‐096‐427, Miltenyi Biotec). The resulting muscle slurry was filtered (100 μm; Cat# 22‐363‐549, Fisher Scientific) and then treated with Red Blood Cell Lysing Buffer (Cat# R7767, Sigma‐Aldrich). Cells were incubated with FITC Rat Anti‐Mouse CD45 (Cat# 561088, BD Biosciences), BV510 Rat Anti‐Mouse CD31 (Cat# 563089, BD Biosciences), anti‐Alpha 7 Integrin (ITGA7) 647 (Cat# 67‐0010‐05, AbLab) and BV711 Rat Anti‐Mouse Ly‐6A/E (Sca1) (Cat# 563992, BD Biosciences) in flow buffer (10% FBS, 3 mM of EDTA in 1× PBS) for 35 min. Cell viability was assessed with SYTOX™ Green (Cat# S34860, Invitrogen). Cell populations of interest were haematopoietic (CD31−/CD45+), endothelial (CD45−/CD31+), MuSCs (CD45−/CD31−/ITGA7+) and FAPs (CD45−/CD31−/ITGA7−/Sca1+). Analysis was performed using an NxT flow cytometer and processed in FlowJo™ v10.8 Software (BD Life Sciences) with gates and compensation established using fluorescence minus one and single‐stained controls.
4
Granulocyte Identification in BALF
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For classification of granulocytes, cells from BALF were stained with Zombie Aqua™ solution for 10 min in the dark, stained for 30 min with antibodies for markers as follows: FITC Rat Anti-Mouse CD45 (#553079, BD, USA), PE/Cyanine7 anti-mouse CD11c (#117317, BioLegend, USA), BB700 Rat Anti-Mouse CD11b (#566416, BD, USA), APC Rat Anti-Mouse Ly-6G (#560599, BD, USA), PE Rat Anti-Mouse Siglec-F (#552126, BD, USA), BV421 Rat Anti-Mouse F4/80 (#565411, BD, USA). Absolute cell counts were calculated on the basis of Precision Count BeadsTM (#424902, BioLegend, USA). Data were collected on a BD Biosciences FACSVerse Flow Cytometer and analyzed using FlowJo software. The Flow cytometry gating strategy is shown in Supplementary Fig. S1F .
5
Investigating MAPK Pathway Signaling
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Antibody of EGF Receptor (#2232), Phospho-BRAF (Ser445, #2696), CRAF (#53745), Phospho-c-Raf (Ser338, #9427), MEK1/2 (#4694), MEK1 (#12671), MEK1 (#2352), MEK2 (#9147), p-MEK1/2 (Ser217/221, #9154), ERK1/2 (#4695), p-ERK1/2 (Thr202/Tyr204, #4370), GST-Tag (#2624), PD-L1 (#13684) were obtained from Cell Signaling Technology (Danvers, MA), BRAF V600E (#A1137-25) from Biovision (San Francisco, USA), and BRAF (#ab33899), Ki67 (#ab16667) obtained from Abcam (Cambridge, USA). β-Actin (#sc47778) provided by Santa Cruz Biotechnology (Santa Cruz, USA), and anti-Flag antibody (#F1804) was obtained from Sigma–Aldrich (St. Louis, USA). FITC Rat Anti-Mouse CD45 (#553079), PerCP-Cy™5.5-CD8a (#551162), APC-NK-1.1 (#550627), PE-CD4 (#557308) obtained from BD (New Jersey, USA), and in vivo anti-mouse PD-L1 (#BE0101) obtained from BioXcel, USA.
6
Comprehensive Flow Cytometry Analysis
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For flow cytometry analysis, whole blood samples were stained following the almost identical protocol already described elsewhere72 (link) by using the following fluorochrome-labeled anti-mouse mAbs: FITC Rat Anti-Mouse CD45; APC Rat Anti-Mouse Ly-6G; PE-CF594 Rat Anti-Mouse Ly-6G and Ly-6C; PE-Cy7 Rat Anti-Mouse CD4; Alexa Fluor 700 Rat Anti-Mouse CD8a; APC-Cy7 Rat Anti-Mouse CD45R; PE Hamster Anti-Mouse CD3e and PerCP-Cy5.5 Rat Anti-Mouse CD335 (BD Biosciences, San Diego, USA). In brief, protected from light, blood was incubated for 15 min at room temperature. The stained samples were then treated with BD FACS lysing solution (BD Biosciences) following manufacturer’s instructions. Thereafter, white blood cells were washed twice with washing buffer (PBS, 1% BSA and 0.1% sodium azide), resuspended in measuring buffer (PBS, 0.1% BSA and 0.1% sodium azide) and measured by flow cytometry using LSR Fortessa (BD Biosciences, San Diego, USA). Plots were compensated and analyzed by FlowLogic Software 7.2.1 (Inivai Technologie, Mentone Victoria, Australia). Gating strategy is shown in Supplemental Fig. 1.
7
Macrophage Activation and Characterization
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All cell culture media, trypsin, fetal bovine serum (FBS), and antibiotic/antimycotic solutions were obtained from Invitrogen (Carlsbad, CA, USA). The L-929 fibroblast (CCl-1), LADMAC macrophage/monocyte (CRL-2420), and C3H/HeJ mouse I-13.35 splenic cell lines (CRL-2471) deficient for TLR-4 were purchased from the American Type Tissue Collection (ATCC) (Manassas, VA, USA). Endotoxin tested (less than 0.1 ng/μg) IFNγ (#I1000) was purchased from US Biological (Salem, MA, USA), the inducible nitric oxide synthase (iNOS) (#2977) antibody from Cell Signaling Technology (Danvers, MA, USA), the anti-monocyte/macrophage antibody (MOMA-2, ab33451) and anti-integrin beta-1 antibody (CD29) (#ab23834) from Abcam (Cambridge, MA, USA), and the fluorescein isothiocyanate (FITC) rat anti-mouse CD44 (#553133), phycoerythrin (PE) rat anti-mouse CD105 (#562759), PE rat anti-mouse Ly-6AE (Sca-1) (#561076), FITC rat anti-mouse CD45 (#553080), FITC rat anti-mouse CD106 (#553332), PE rat anti-mouse CD73 (#557041), and FITC rat anti-mouse CD11b (#553310) were purchased from BD Biosciences (San Jose, CA, USA). Human ox-LDL was purchased from Intracel (Frederick, MD, USA). Gamma-irradiated LPS from Escherichia coli (#L4391) and all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
8
Immunofluorescent Staining of Cryosectioned Liver Tissue
9
Xenograft Modeling of Leukemia in NSG Mice
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NSG (NOD/Shi-scid/IL-2Rγnull) mice (in-house breeding; female, 4–6 weeks) were sublethally irradiated (250 rads), and each mouse was injected with 0.6 × 106 cells via lateral tail vein. Engraftment was assessed by flow cytometric measurement of human and mouse CD45 expression on the cell surface (APC mouse anti-human CD45 and FITC rat anti-mouse CD45, BD Biosciences). 15 mice were transplanted with MV4;11-C cells, 7 of which were assessed for engraftment. 10 mice were transplanted with MV4;11-KD cells, 5 of which were assessed for engraftment. For REH-C and REH-KD cells, 5 mice were transplanted with each cell line, and all were assessed for engraftment. The sample sizes were chosen based on efficiency of resource use, past experience with similar experiments, and pilot experiments to determine the effect size. No animal was excluded from the analysis, randomization was not used, and the investigator was not blinded. All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD, USA) and were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.
10
Xenograft Modeling of Leukemia in NSG Mice
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