BM cells or splenocytes were harvested and subjected to red blood cell lysis. Fresh or frozen cells were stained with the following Abs: CD45.2-FITC and CD45.1-APC, Mac1-PE, Gr1-APC, c-Kit–APC, CD71-PE, Ter119-APC, B220-PE, and CD3-APC (BD) and analyzed on the BD FACSCalibur instrument. Staining for multiparameter flow cytometry was performed after a c-kit enrichment using 10 µl MACS beads (CD117) per mouse and then run on an AutoMACS (Miltenyi Biotec) according to the manufacturer’s instructions. The cells were then stained with the following cocktail: (Lineage; CD3, CD4, CD8, Gr1, B220, CD19, and TER119 all conjugated with PeCy5), Sca-Pac Blue, CD34-FITC or CD45.2-FITC, SLAM-APC, CD48-PE, c-KIT–Alexa Fluor 780, and FcgRIIb-PeCy7 (Fig. 2 d ); HPCs (Linloc-Kit+Sca1−), GMPs (LK, FcγRIIbhiCD34+), CMPs (LK, FcγRIIbmid CD34+), MEPs (LK, FcγRIIbloCD34−), B cells (B220+), and T cells (CD3+) from the spleen were also sorted. For analysis of LMPPs and CLPs, the following cocktail was used: Lineage marker mix–PeCy5, Sca-Pecy7 IL-7Ra–Pac Blue, Flk2-PE, CD34-FITC, and Kit-APC (Martin et al., 2003 (link); Adolfsson et al., 2005 (link); Karsunky et al., 2008 (link)).
Gr1 apc
Manufactured by BD
Sourced in United States
Gr1-APC is a flow cytometry reagent produced by BD that is used to detect the Gr1 antigen, which is expressed on the surface of granulocytes, monocytes, and myeloid-derived suppressor cells. It is a fluorescently-labeled monoclonal antibody that can be used in flow cytometric analysis to identify and quantify these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b fitc, by BD (4 mentions)
Cd3 apc, by BD (3 mentions)
Cd4 fitc, by BD (3 mentions)
Cd45 percp cy5, by BD (3 mentions)
Cd45 pe cy7, by BD (3 mentions)
Cd11b apc cy7, by BD (3 mentions)
F4 80 pe cy7, by BD (3 mentions)
Fc block, by BD (3 mentions)
Cd3 fitc, by BD (3 mentions)
Cd71 pe, by BD (2 mentions)
Cd25 fitc, by BD (2 mentions)
Cd8a fitc, by BD (2 mentions)
Cd45 pe, by BD (2 mentions)
Cd45 bv421, by BD (2 mentions)
Cd11b pe, by BD (2 mentions)
Cd4 v500, by BD (2 mentions)
Cd11b percp cy5, by BD (2 mentions)
Nk1.1 apc, by BD (2 mentions)
Cd3 eflour450, by Thermo Fisher Scientific (2 mentions)
Ter119 apc, by Thermo Fisher Scientific (2 mentions)
17 protocols using gr1 apc
1
Multiparametric Flow Cytometry Analysis of Hematopoietic Cells
2
Immune Profiling of Tumor-Bearing Mice After MVA-TWIST/TRICOM Vaccination
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Expression of B7-1, ICAM-1, and LFA-3 was determined by flow cytometry. Treated MC38 cells were stained with FITC-labeled antibodies to CD80 (B7-1), CD54 (ICAM-1) and CD48 (LFA-3) (BD Biosciences, San Jose, CA). Cells were incubated with antibodies for 30 min at 4°C. Samples were acquired on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The effect of MVA-TWIST/TRICOM on splenic immune cell populations was examined in tumor bearing and non-tumor bearing BALB/c mice 17 days after receiving two vaccinations with MVA-TWIST/TRICOM or being left untreated (n = 5/group). Vaccination of tumor bearing mice began 4 days post-implantation of 5 × 104 4T1 mammary tumor cells. Spleens were prepared and stained as described previously [60 (link)], using the following antibodies: CD3e-V500, t-APC, CD8a-Pacific Blue, CD25-FITC, CD44- PerCP-Cy5.5 CD11b-V500, Gr-1-APC, CD11c-PerCP-Cy5.5, CD40-FITC (BD Biosciences); CD62L-PE-Cy7, FoxP3-PE, MHC II-efluor450 (eBioscience, San Diego, CA); and CD49b-PE-Cy7 (Biolegend, San Diego, CA). Tetramer staining (Beckman Coulter, Pasadena, CA) was performed on splenocytes from non-tumor bearing mice following 7 days of in vitro stimulation with 1.0 μg/mL Twist peptide (BALB/c - LYQVLQSDEL). All samples were acquired on a BD Verse flow cytometer. All marker expression was determined using FlowJo software (TreeStar, Inc., Ashland, OR).
3
Immunomodulation of Tumor Microenvironment
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CpG ODN 1826 (5ʹ-tccatgacgttcctgacgtt-3ʹ) and isotype control were purchased from InvivoGen (San Diego, CA, USA). Anti-OX40 (CD134) monoclonal antibody (clone OX86) and isotype control were purchased from BioXCell (Lebanon, NH, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin solution, phosphate-buffered saline (PBS) and TRIzol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Matrigel was purchased from Corning (Corning, NY, USA). The TUNEL Apoptosis Detection Kit was purchased from KeyGen BioTECH (Nanjing, Jiangsu, China). The T Cell Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies including CD45-PerCP-Cy5.5, CD3e-BV510, CD4-FITC, CD25-PE, OX40-APC, CD8a-FITC, CD11b-BV421, Gr-1-APC, F4/80-PE, CD8a-APC, CD44-PE-Cy7, CD62L-PE, IFN-γ-PE, and FoxP3-eFluor450 were purchased from BD Biosciences (San Jose, CA, USA) and Biolegend (San Diego, CA, USA).
4
Comprehensive Immune Cell Profiling
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Monoclonal antibodies CD3e-ApcCy7, CD45-PerCPCy5.5, Gr1-APC, CD8-PECy7, CD25-FITC, CD11b-FITC, CD11C-PE, B220-PB, and NK1.1-PE were purchased from BD Biosciences. Foxp3-APC and F4/80-PE were purchased from eBioscience and CD4-PO and calcein violet from Invitrogen.
5
Multiparametric Flow Cytometry Analysis
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Antibodies against cell surface marker and detect intracellular pathways were purchased from BD Pharmingen (San Jose, CA, USA): CD11b-FITC, Gr-1-APC, FITC-CD3, PE-CD28, FITC-CD25, PE-Foxp3, APC-CD4, APC-IFN-γ, purified anti-CD16/CD32 (mouse BD Fc BlockTM, BD Biosciences, Franklin Lakes, New Jersey, USA).
6
Flow Cytometry Analysis of Tumor Infiltrates
7
Tumor Single-Cell Isolation and Immune Profiling
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After intracardiac injection of PBS, breast cancer tissues, lung metastases or lungs were harvested, minced and digested at 37°C for 45 min with DMEM medium containing collagenase type 1A (1.5 mg/ml), hyaluronidase (1.5 mg/ml), and DNase (20 U/ml). The digestion mixtures were filtered through 70 μm cell strainers. Single-cell suspensions were incubated with rat anti-mouse CD16/CD32 mAb (BD Biosciences), and then stained, washed and re-suspended in cold buffer (1%BSA, 0.1% NaN3 in PBS). 7AAD reagent (eBioscience) was added to the stained tubes (5 μl/tube) just before running the flow analysis. Flow cytometry data were acquired on a Gallios flow cytometer (Beckman, USA), and data were analyzed with Kaluza software (version 1.3). The appropriate, fluorochrome-conjugated, isotype-matched, control IgGs were used in all experiments. The following monoclonal anti-mouse antibodies were used: CD45-PE-Cy7, CD45-PerCP, CD45-BV421, Gr1-PerCP-Cy5.5, Gr1-APC, Gr1-APC-Cy7, CD11b-APC-Cy7, CD11b-BV510, Ly-6G-FITC, Ly-6C-PE (BD Biosciences) and F4/80-PE, F4/80-FITC, F4/80-APC (eBioscience).
8
Flow Cytometry Analysis of Immune Cells
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Human peripheral blood mononuclear cells, human peritoneal effluent cells and murine peritoneal effluent cells were analyzed by flow cytometry.
Cells were isolated from PD effluents by centrifugation at 500 g for 15′ at 4°C. PBMCs from healthy control donors were prepared by Ficoll-Hypaque (Amersham Pharmacia Biotech, Sweden) density gradient centrifugation. The following mAb were used for flow cytometry analysis of surface molecules: CD3-PerCP, CD14-FITC, CD56-APC (BD Biosciences) and Fn14-PE (eBioscience). Cells were pre-incubated with 50 µg/mL human IgG to prevent binding to FcR and stained according to standard protocols. Analysis was performed in a FACScalibur cytometer with ProQuest software (BD Bioscience).
Cells from murine peritoneal effluent were analyzed by flow cytometry using the following antibodies: anti-mouse CD4 PE and Alexa Fluor® 488-labelled anti-mouse CD8a for T cells. F4/80 PE-Cy7, Gr1 APC, CD14 FITC and CD11b PE for macrophages (BD Biosciences, San Diego, USA). Macrophages were defined as CD11b+F4/80+ cells and Gr1+ macrophages as CD11b+F4/80+Gr1+ cells. Neutrophils were defined as Gr1+/F4/80- cells.
Cells were isolated from PD effluents by centrifugation at 500 g for 15′ at 4°C. PBMCs from healthy control donors were prepared by Ficoll-Hypaque (Amersham Pharmacia Biotech, Sweden) density gradient centrifugation. The following mAb were used for flow cytometry analysis of surface molecules: CD3-PerCP, CD14-FITC, CD56-APC (BD Biosciences) and Fn14-PE (eBioscience). Cells were pre-incubated with 50 µg/mL human IgG to prevent binding to FcR and stained according to standard protocols. Analysis was performed in a FACScalibur cytometer with ProQuest software (BD Bioscience).
Cells from murine peritoneal effluent were analyzed by flow cytometry using the following antibodies: anti-mouse CD4 PE and Alexa Fluor® 488-labelled anti-mouse CD8a for T cells. F4/80 PE-Cy7, Gr1 APC, CD14 FITC and CD11b PE for macrophages (BD Biosciences, San Diego, USA). Macrophages were defined as CD11b+F4/80+ cells and Gr1+ macrophages as CD11b+F4/80+Gr1+ cells. Neutrophils were defined as Gr1+/F4/80- cells.
9
Analysis of FOXO and Cell Cycle Markers
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CD34+ cells were resuspended in “Fix and Perm” (Merck Chemicals, Ltd., Nottingham, U.K.). Primary antibodies, FOXO1, FOXO3a, FOXO4, p-FOXO1 (Ser319), p-FOXO3a (Ser253), p-FOXO4 (Ser193), and Cyclin D1 (Cell Signaling), added at RT for 1 hour and secondary for 30 minutes. Cell cycle was analyzed with PI (50 µg/ml) (Sigma-Aldrich Company, Ltd., Dorset, UK), DAPI, 7-AAD, and Ki67 (BD Biosciences, Oxford Science Park, U.K.). Surface markers Annexin V-FITC, CD34-APC, Mac1(CD11b)-PE, and Gr1-APC (BD Biosciences) were added at RT for 15 minutes.
10
Comprehensive BMDM Phenotyping and Sorting
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Entire cell populations of day 4 BMDMs were stained with a mix of monoclonal antibodies CD11b–Alexa 700, Gr-1–APC (The Walter and Eliza Hall Institute Monoclonal Antibody Facility), and CD115-PE (BD) in 1 ml FACS buffer (PBS, 2% FCS, and 2 mM EDTA) for 20 min on ice and sorted with a cell sorter (Aria W; BD). Flow cytometric analysis of hematopoietic cells was performed with a cell analyzer (LSRFortessa; BD), and data were analyzed and processed using FlowJo software. Sorted cells were cytocentrifuged at 1,000 rpm for 5 min, stained using a May-Grünwald Giemsa solution, and inspected by light microscopy.
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