The following antibodies were used in this study: anti-Ly6A/E (Sca1) (D7), anti-CD48 (HM48-1), anti-CD41 (MWReg30), anti-CD45.2 (104), anti-CD45.1 (A20), anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti-B220 (RA3-6B2), anti-Ki-67 (SolA15), anti-FcRII/III (93) and anti-Ter119 (TER-119) were all from eBioscience. Anti-CD150 (TC15-12F12.2), anti-CD117 (2B8), anti-CD11b (M1/70), anti-CD31 (MEC13.3), anti-CD144 (BV13) and anti-CD34 (MEC14.7) were from Biolegend. Anti-Lineage panel cocktail (TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11 at 1:50 dilution) was from BD Biosciences. Streptavidin eFluor 450 and APC eFluor 780 from eBioscience and streptavidin BV 570 from Biolegend were used for biotinylated antibodies. Unless otherwise specified, all antibodies and streptavidin cocktails were used at a 1:100 dilution.
Rb6 8c5
Manufactured by BD
Sourced in United States
The RB6-8C5 is a laboratory equipment designed for use in scientific research and analysis. It serves as a device for controlled liquid handling and sample preparation. The core function of this product is to provide precise and accurate liquid dispensing and aspiration capabilities to support various laboratory workflows.
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Lab products found in correlation
M1 70, by BD (5 mentions)
Ra3 6b2, by BD (3 mentions)
Ter119, by BD (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
E50 2440, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
B220 ra3 6b2, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Annexin 5 pe, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Anti ter119 ter 119, by Thermo Fisher Scientific (1 mentions)
Streptavidin efluor 450, by Thermo Fisher Scientific (1 mentions)
Anti cd144 bv13, by BioLegend (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Tcrβ h57 597, by BD (1 mentions)
Cd45.2 104, by BD (1 mentions)
Cd4 gk1, by BD (1 mentions)
4 6 diamidino 2 phenylindole dapi solution, by Dojindo Laboratories (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
23 protocols using rb6 8c5
1
Comprehensive Immune Cell Profiling
2
Competitive Bone Marrow Repopulation Assay
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Competitive repopulation assays were performed by injecting 2.5×106 bone marrow cells from either experimental (Mdm2C305F/C305F) or control (Mdm2C305F/+, C57BL/6) donor animals on a CD45.2 background, along with 2.5×106 competitor cells from CD45.1 C57BL/6 mice into irradiated (900 cGy) C57BL/6 recipient mice (CD45.1) [26 (link), 27 (link)]. Peripheral blood samples were collected at 4, 8, and 12 weeks following transplant to assess for the percentage of reconstitution of peripheral blood cell lineages with CD45.2 cells, thus defining relative repopulation capacity of bone marrow harvested from our experimental versus control mouse strains in each lineage analyzed. Leukocyte subsets were assessed after RBC lysis with ammonium chloride, using the following antibodies: FITC antibodies against, B220 (RA3-6B2, BD Biosciences), TCR β (H57-597, BD Biosciences), CD45.2 (104, BD Biosciences), and CD4 (GK1.5, BD Biosciences); PE antibodies against, CD11b (M1⁄70, BD Biosciences), Gr-1 (RB6-8C5, BD Biosciences), CD8 (53–6.7, eBioscience), and CD45.1 (A20, BD Biosciences).
3
Immunofluorescence Staining of Mouse Tumors
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Mouse tumour cryosections (6 μm) were stained with anti-CD8 (Abcam, clone YTS169.4), anti-Gr-1 (BD Pharmingen, clone RB6-8C5) and anti-CD31 (BD Bioscience, clone MEC 13.3) antibodies as described.9 (link) HypoxyprobeTM-1 OMNI kit (HPI Inc., Burlington, USA) was used for detection of hypoxic area in mouse tumour samples following the manufacturer's protocol. Image J software was used for quantification of CD31-positive area and pimonidazole-positive area in tumour. For immunofluorescence staining, anti-IgG labelled with AlexaFluor 488 (Abcam, Kenbridge, UK) and AlexaFluor 594 (Abcam) were used as secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution (Dojindo, Kumamoto, Japan). Fluorescence images were captured using a Keyence BZ-X700 microscope (Keyence, Osaka, Japan).
4
MDSC Isolation, Culture, and Analysis
5
Single-cell RNA-seq of Myeloid Cells
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Cells were prepared as described in the flow cytometry section. After blocking, cells were spun and incubated with the following antibodies in 1% BSA for 30 minutes: BV786 rat anti-mouse CD45 (Catalog #: 557659, 30-F11, BD Pharmigen), eFluor®450 rat anti-mouse F4/80 (Catalog #:48-4801, BM8, eBioscience), APC rat anti-mouse CD11b (Catalog #:17-0112, M1/70, eBioscience), APC-Cy7 rat anti-mouse Gr-1 (Catalog #:557661, RB6-8C5, BD Pharmingen), and Fixable Aqua Dead Cell Stain (catalog no. L34957; Invitrogen). After staining for 30 minutes, cells were spun, washed with 1% BSA, and sorted using a Becton–Dickenson FACSAriaII. We sorted ~25,000 live CD11b+ and F4/80+ cells from individual animals (n=2 per group) into individual BSA coated tubes. Cells were counted and approximately 5,000 cells from each animal were subjected to 10X genomics. This was done for n=2 animals from each experimental group; all animals were female.
6
Intravital Lung Microscopy Protocol
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Intravital microscopy of the lung was performed as described previously8 (link)66 (link). Briefly, animals were anaesthetized and mechanically ventilated. A thoracotomy was performed to expose the right middle lung lobe and the lung was hold in position using a custom-built fixation device with an integrated observation window66 (link). Immediately before imaging animals were injected with an Alexa488-coupled anti-Gr1 antibody (clone RB6-8C5, 5 μg per mouse, purified from hybridoma supernatant) and a PE-coupled anti-CD41 antibody (clone MWReg30, 5 μl per mouse, BD Biosciences, Franklin Lakes, NJ, USA). High-speed multichannel fluorescence microscopy was performed on an upright microscope (Axioskop; Carl Zeiss, Göttingen, Germany) equipped with a Lambda DG-4 ultra high speed wavelength switcher (Sutter Instruments, Novato, CA, USA) and a 40 × 0.75 NA saline immersion objective. Videos were recorded with a digital camera (Sensicam QE) and analysed with Slidebook Software (Version 5; Intelligent Imaging Innovations, Göttingen, Germany).
7
Multicolor Immunofluorescence Characterization of Liver Cells
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Indirect immunofluorescence was performed on frozen and fixed (acetone:methanol) liver tissue using monoclonal antibody CD45-FITC (1:100) (30-F11, BD Pharmingen, San Jose, USA). Co-localisation was assessed using CD147 (1:100) (RL73.2, hybridoma), Gr-1 (1:100) (RB6-8C5, BD Pharminogen) and F4/80 (1:200) (CI:A3-1, hybridoma), B220 (1:100) (RA3-6B2, BD Phaminogen), CD19 (1:100) (ID3, BD Pharmingen) and CD3e (1:100) (UCHT1, Dako). Secondary antibodies anti-rat AlexaFluor® 594 (1:200) (#A-11007, Molecular Probes) and goat anti-rabbit AlexaFluor® 633 (1:200) (#A-21071, Invitrogen) were used. For control of background staining, the primary antibodies were omitted or replaced by IgG isotype control (Cat ID: 559073, Dako, San Clara, USA). The fluorescent images were imaged using a Leica TCS SP5 II confocal microscope (Leica, Georgia, USA).
8
Murine Brain Cell Isolation and Flow Cytometry
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Single-cell suspensions were obtained from brain tissues by mechanical isolation. Extracellular myelin was removed from all brain and spinal cord suspensions using myelin removal beads (Miltenyi Biotec), according to the manufacturer’s instructions. For surface staining, cell suspensions were blocked with 1% anti-mouse CD16/CD32 (eBioscience; 93) for 20 min on ice and then stained with fluorescently labeled antibodies for murine CD4 (BD Biosciences; RM4-5), CD11c (eBioscience; N418), CD11b (eBioscience; M1/70), or GR1 (BD Biosciences; RB6-8C5), for 45 min. Following staining, cells were washed with PBS, lysed for red blood cell (Beckman Coulter), and fixed using a fixative agent (eBioscience). All samples were analyzed or sorted on a BD FACSAria II (BD Bioscience). Data were analyzed using the FlowJo 10 Data Analysis software (FlowJo LLC) and BD FACSDiva software (BD Biosciences).
9
Isolation and Transcriptomic Analysis of Murine Gr-1+ Cells
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Gr-1+ (Ly6G+) cells were isolated from splenocytes of host mice by magnetic-activated cell sorting (MACS) using anti-Ly6G and anti-Ly6C Abs (RB6-8C5; BD Pharmingen, San Jose, CA, USA) on day 7 after allo (or syngeneic) mice. Cells were used for RNA extraction when the purity was higher than 97%. For microarray hybridization, total RNA was isolated and purified using a DNA-free RNA isolation kit (RNAqueous-4PCR kit; Ambion, Austin, TX, USA) according to the manufacturer's instructions. RNA samples were first amplified for array analysis using the Illumina Total Prep RNA Amplification Kit (Ambion) as previously described (29 (link)). Amplified cRNA (1.5 µg) was hybridized on Mouse WG-6 v2 Expression BeadChip arrays (Illumina Inc., San Diego, CA, USA), containing more than 45,281 well-annotated Ref transcripts. The array chip was then scanned on a BeadArray Reader (BeadStation 500G Instrument; Illumina Inc.) and subjected to gene identification and quantitation by the Genome Studio Software v1.0.2. (Illumina Inc.).
10
Neutrophil Depletion in Mice for Tissue Stimulation Studies
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In some experiments, mice were depleted of their circulating neutrophils before tissue stimulation as previously described (Voisin et al., 2009 (link)). In brief, anti-GR1 antibody (25 µg/day; RB6-8C5; BD) in 500 µl saline was injected i.p. for 3 consecutive days. Control mice received rat IgG2bκ isotype control antibody (AbD Serotec). The level of neutrophils and monocytes was determined by flow cytometry (Gr1+ and Gr1+/CX3CR1-GFP+ cells, respectively) from blood samples taken from the tail vein before and after antibody treatment. Mice treated with anti-Gr1 antibody showed >85% depletion of their circulating neutrophils with no impact on the proportion of blood Gr1+/CX3CR1-GFP+ monocytes.
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