Macrophages from peritoneal exudate derived from mice in the TGC model (0.5 × 106 cells) were resuspended in FACS buffer (PBS supplemented with 0.5% bovine serum albumin and 0.1% NaN3) and were labeled with the antibody mixes from panel 1 (CD45-APC; F4/80-Viogreen; MHCII-Vioblue; LFA-1-PeVio770; CD64-PE; CD16/32-Viobright FITC) (Miltenyi Biotec, Bergisch Gladbach, Germany) and panel 2 (CD45-APC; F4/80-Viogreen; CD64-PE; MHCII-Vioblue; CD38-PeVio770; CD206-FITC) (Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 min at 4°C. Cells were then washed with FACS buffer and the relative fluorescence intensities were determined using a MACSQuant Vyb cytometer equipped with FlowLogic software (Miltenyi Biotec, Bergisch Gladbach, Germany).
Cd45 apc
Manufactured by Miltenyi Biotec
Sourced in Germany
CD45-APC is a fluorescently labeled antibody that binds to the CD45 antigen, which is expressed on the surface of most leukocytes. It can be used in flow cytometry applications to identify and quantify different types of white blood cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by Miltenyi Biotec (4 mentions)
Cd8 vioblue, by Miltenyi Biotec (3 mentions)
Cd14 viogreen, by Miltenyi Biotec (3 mentions)
Cd4 pe, by Miltenyi Biotec (3 mentions)
Cd20 percp, by Miltenyi Biotec (3 mentions)
Cd56 apc vio770, by Miltenyi Biotec (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Cd11b fitc, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cd3 percp vio700, by Miltenyi Biotec (2 mentions)
D luciferin, by Ozyme (2 mentions)
Caliper ivis lumina 2 system, by PerkinElmer (2 mentions)
Cd45ra percpvio770, by Miltenyi Biotec (2 mentions)
Cd62l pe, by Miltenyi Biotec (2 mentions)
Macsquant 10, by Miltenyi Biotec (2 mentions)
Cd34 pe, by Miltenyi Biotec (2 mentions)
Flowlogic software, by Miltenyi Biotec (1 mentions)
Mhcii vioblue, by Miltenyi Biotec (1 mentions)
Cd206 fitc, by Miltenyi Biotec (1 mentions)
Macsquant vyb cytometer, by Miltenyi Biotec (1 mentions)
17 protocols using cd45 apc
1
Characterizing Murine Peritoneal Macrophages
2
Microglia Isolation from P0-3 Mouse Brains
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Brains of P0-3 pups from C57BL/6 mice were used to isolate microglial cells. A single cell suspension was prepared using papain based neural tissue dissociation protocol (Miltenyi Biotec, 130-092-628). Primary microglia were isolated using CD11b Microbeads (Miltenyi Biotec GmbH, 130-093-634) according to the manufacturer’s instruction. The purity of the isolated cells was determined by staining with fluorescently labelled antibodies APC-CD45 and FITC-CD11b (Miltenyi Biotec, 130-091-811 and 130-081-201) and analyzed by Flow Cytometry (purity was approximately 90%). Microglia cells were re-suspended in DMEM/F12 (Gibco, 11320-074) supplemented with 10% FBS (Gibco, 10082139), 0.1% nonessential amino acids (Gibco, 11140-050), 0.1% GlutaMAX (Gibco, 35050-038) and 1% Penicillin/Streptomycin and plated into 96 well plates (2×104 cells/well) for cytokine release using Meso Scale Discovery (MSD) or into 12-well plates for RNA isolation.
3
Characterization of Lung Immune Cells
4
Melatonin Enhances Anti-Tumor Immunity
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All animals were cared for in a specific pathogen-free room and treated following the animal care protocol approved by an animal committee. Mouse LLC1 (1 × 106) cells were subcutaneously injected into 8-week-old -C57BL/6 mice. After one week of inoculation, the mice were intraperitoneally administered melatonin (30 mg/kg, three times per week) for 4 weeks. The tumor volume and mice body weights were monitored every 2 days during the experiments. Tumor volume was calculated using the following equation: length × (width)2 × 0.5. To analyze tumor-infiltrating lymphocytes (TILs), freshly isolated tumor tissue was cut into small pieces and disassociated by the gentleMACS tumor dissociation kit (Miltenyi Biotec). The suspension was further treated with a RBC lysis buffer to remove red blood cells. Approximate 1 × 106 isolated cells were incubated with fluorophore-conjugated antibodies including CD45-APC, CD11b-PE, CD3-FITC, CD8-APC-Vio770, Ly6G-FITC, CD4-PE-Vio770, and F4/80-APC (Miltenyi Biotec) and analyzed by the BD FACSVia flow cytometer (BD Biosciences) [43 (link)].
5
Xenograft Mouse Model for CAR T Cell Therapy
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Animal studies were approved by the state animal research committee (LANUV, NRW, Germany) and all animals were cared for according to the guidelines set by the Federation of European Laboratory Animal Science Associations. Six- to 8-week-old female NOD.Cg-PrkdcSCIDIl2rgtm1Wjl/SzJ (NOD-SCID gamma; NSG) mice (Charles River Laboratories, Sulzfeld, Germany) were intravenously engrafted with 3.5 × 106 MOLM-14 cells stably expressing a firefly luciferase-EGFP fusion protein (LucEG). Six days later, mice were intravenously injected with 3.5 × 106 N3-, N4-, or CD8-hinged CD19 or CD33 CAR T cells. At days 6, 13, 20, 27, and 34, the persistence of MOLM-14 cells was assessed via luminescence imaging and PB analysis. For luminescence imaging, mice were intraperitoneally injected with D-luciferin (OZ Biosciences SAS, Marseilles, France) and after 5 min luminescence was measured in a Caliper IVIS Lumina II system (PerkinElmer LAS, Rodgau, Germany) with an exposure time of 15 s. PB was drawn from the tail vein, the erythrocytes lysed with BD Pharm Lyse (BD Biosciences), and the samples analyzed on a MACSQuant Analyzer X flow cytometer for EGFP, CD33, and CD45 expression for MOLM-14 cells and BFP, CAR (ΔNGFR), CD3, and CD45 expression for CAR T cells after staining with CD271-PE, CD3-PerCP-Vio700, CD45-APC, and CD33-APC-Vio770 (all from Miltenyi Biotec)
6
Xenograft Tumor Injection and PBMC Isolation
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K562-luc2 cells were thawed from cryopreservation 48h before tumor injections and maintained at 37°C, 5% CO2 in Iscove’s DMEM supplemented with 10% FBS and 8µg/mL blasticidin for 2 days. On the day of tumor injections, K562-luc2 cells were removed from culture, washed three times with sterile PBS, and resuspended at a concentration of 1x106 cells/200µL in sterile saline. Cryopreserved PBMCs were thawed in a 37°C water bath for two minutes or until a small ball of ice remained in solution. PBMCs were resuspended in 5mL RPMI + 10% FBS. A small aliquot (50 µL) was taken, and cells were labeled with monoclonal antibodies (CD8-VioBlue, CD14-VioGreen, CD3-FITC, CD4-PE, CD20-PerCP, CD45-APC, CD56-APC-Vio770 (Miltenyi Biotec) for surface staining prior to injection. The remaining cells were supplemented with IL-15 (0.1mg/mL) and incubated for 1 hour at 37°C, 5% CO2 to improve activation and recovery of NK cells functions. After incubation, PBMCs were washed three times with PBS to remove all RPMI media and then resuspended at a concentration of 10x106 PBMCs/200µL sterile filtered saline for injections.
7
Characterization of Naepcs and Adv Receptors
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NAEPCs were characterized through flow cytometry using CD45-APC (Miltenyi Biotec, Germany), CD326 (EpCAM)-PE (Miltenyi Biotec, Germany), and anti-cytokeratin-FITC (Miltenyi Biotec, Germany), followed by fixation and permeabilization using Inside Stain Kit (Miltenyi Biotec, Germany) according to manufacturer’s instructions. The selected antibodies for the characterization of NAEPCs were adapted in a previously published study [20 (link)]. The AdV receptors, CD46-APC (Miltenyi Biotec, Germany), CXADR/CAR-PE, and DSG-2-PE (Affymetrix, Thermo Fisher Scientific) were chosen for flow cytometry analyses.
8
Isolation and Purification of EpCAM+ Cells
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Fresh pleural or peritoneal punctates were processed immediately. The whole cellular fraction was frozen and stored or immediately depleted from most CD45+ cells using magnetic human CD45 microbeads (Miltenyi Biotec 130-045-801). Cells were stained with EpCAM-FITC (Miltenyi Biotec) and CD45-APC (Miltenyi Biotec) in suspension or further purified by sorting of singlet-gated EpCAM+CD45−DAPI− cells on a FACSAriaTM Fusion device (BD Biosciences) and fixed in 1.6% PFA. Cells were stained as indicated, attached to microscopy slides and imaged on a Leica TCS SP8 confocal microscope.
9
Multiparametric Flow Cytometry of Mesenchymal and Stem Cells
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Detached cells were briefly resuspended in 200 µL of FACS buffer (1% FBS in PBS) and incubated with antibodies for 30 min. We used CD29-APC, CD44-APC, CD90-APC, CD105-APC as MPC markers, and CD34-APC, CD45-APC, SSEA4-APC, TRA-1-60-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) as hematopoietic and stem cell markers. Cells were washed twice with FACS buffer and analyzed using a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA).
10
Microglia Isolation and Characterization
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Single cell suspension from fresh brain tissue was created using Adult Brain Dissociation Kit (Milteny Biotec, 130-107-677) and the GentleMACS Milteny Biotec), as described before [7, (link)31] according to the manufacturer´s protocol. Afterwards, the cells were resuspended in FACS staining buffer (PBS 1X + 1% FCS + 0.1% Na-Azide) and stained for 30 min against mouse CD11b-PerCP-Vio700 Clone REA592 (1:50), CD45-APC (1:50), CD68-PE Clone REA835 (1:50) (Milteny Biotec) in a 96-well plate. The flow cytometry was conducted using BD ® LSR II Flow Cytometer and analyzed with FlowJo Software (version 10.8.1). Briefly, in the FACS analysis, forward scatter (FSC) and side scatter (SSC) parameters were used to select the region of interest (ROI), focusing on single cells and excluding doublets to capture individual microglia populations. Within these ROIs, cells were analyzed for CD11b and CD45 expression to distinguish microglia (CD11b + /CD45 low ) from other monocytes (minimum number of cells: 100.000). Microglia were further analyzed for CD68 expression, a marker for reactive microglia.
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