To optimize the dosing regimen for CD8 T cell depletion, C57BL/6J mice received 300 μg of anti‐CD8α mAb (Bio X Cell Cat#BE0061, RRID:AB_1125541) or isotype control (Bio X Cell Cat# BE0090) mAb via the intraperitoneal route on days −3, −1, 6, 13, and 20. Spleens were harvested on days 5, 10, 17, and 25 and single‐cell suspensions were stained with APC‐H7 rat anti‐mouse CD8α (BD Biosciences Cat#560182, RRID: AB_1645237), PerCP/Cy5.5 rat anti‐mouse CD45 (BD Biosciences Cat# 561869), and PE rat anti‐mouse CD4 (BD Biosciences Cat# 553049). To confirm CD8 T cell depletion following ABLV‐luc infection, spleens were harvested from an isotype control mouse and a CD8 T cell‐depleted mouse on day 5 post‐infection. Single‐cell suspensions were stained with Alexa Fluor 647 rat anti‐mouse CD8α (clone 53‐6.7, purified from hybridoma supernatants and labeled in‐house), PerCP/Cy5.5 rat anti‐mouse CD45 (BD Biosciences Cat# 561869), and PE rat anti‐mouse CD4 (BD Biosciences Cat# 553049). Flow cytometry was performed with a BD LSRII flow‐cytometer, and data were analyzed with FlowJo Software.
Percp cy5.5 rat anti mouse cd45
Manufactured by BD
Sourced in United States
PerCP-Cy5.5 Rat Anti-Mouse CD45 is a fluorescently-labeled monoclonal antibody that binds to the CD45 antigen expressed on mouse leukocytes. It is used for the identification and enumeration of mouse leukocyte populations in flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (2 mentions)
Pe cy7 rat anti mouse cd31, by BD (2 mentions)
Ebioscience fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Brilliant violet 421 anti mouse cd326 ep cam, by BioLegend (2 mentions)
Anti mouse cd16 cd32, by BD (2 mentions)
Pe rat anti mouse cd4, by BD (1 mentions)
Fitc hamster anti mouse cd3e, by BD (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Apc rat antimouse cd4, by BD (1 mentions)
Pe rat anti mouse cd8a, by BD (1 mentions)
Fixable viability stain 780, by BD (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsaria fusion flow cytometer, by BD (1 mentions)
Dnase, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Diva 8, by BD (1 mentions)
L cysteine, by Merck Group (1 mentions)
8 protocols using percp cy5.5 rat anti mouse cd45
1
Optimizing CD8 T Cell Depletion in Mice
2
Apoptosis and Immune Profile Analysis in MCF-7 Cells
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The cell apoptosis state of MCF‐7 cells on scaffolds was carried out by an Annexin V‐FITC/PI assay kit (Neobioscience, China). The cells were collected and stained with Annexin V‐FITC and PI in binding buffer. For evaluating HIF‐1α expression, cells were treated by 2% formaldehyde, 0.1% Triton X‐100, and anti‐HIF‐1α (Cell signaling technology, USA) for 30 min away from light. For immune cells analysis, a single cell suspension (100 µL, 5×107 cells mL−1) of dissociated tumor tissue was seeded on the scaffolds and cultured for 48 and 72 h. The cancer cells were harvested, rinsed with PBS, and resuspended. Then, the collected cells with Fixable viability stain 780, PerCP‐Cy5.5 Rat Antimouse CD45, FITC Hamster Antimouse CD3e, APC Rat Antimouse CD4, and PE Rat Antimouse CD8a (BD Biosciences, USA) were incubated at 4 °C for 30 min away from light. The tests were carried out on a FACS Celesta flow cytometer (BD Biosciences, USA) and calculated the data with FlowJo 10.
3
Isolation of Lung Cell Populations
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Single cell suspensions of whole lungs were prepared as described previously [83 (link)]. For FACS, single cell suspensions were incubated with eBioscience Fixable Viability Dye eFluor™ 780 (Invitrogen) and the following antibodies (0.6 μg per 107 cells): PerCP-Cy5.5 Rat Anti-Mouse CD45 (BD Pharmingen, 550994), PE-Cy7 Rat Anti-Mouse CD31 (BD Pharmingen, 561410), and Brilliant Violet 421 anti-mouse CD326 (Ep-CAM) (BioLegend, 118225) on ice for 45 min, and then subjected to FACS-sorting using FACS Aria II (BD Biosciences) at the Moody Foundation Flow Cytometry Core Facility at the Children’s Research Institute at UTSW. Flow cytometry data were analyzed with FlowJo v10.
4
Flow Cytometry Analysis of siRNA Transfection
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MH-S cells were seeded in 12-well plates and allowed to settle overnight. Particles were prepared as described above and diluted in Opti-MEM (final NG concentration of 30 µg/mL; final siRNA concentration of 100 nM) before incubation with the cells (4 h at 37 °C and 5% CO2). Afterwards, the cells were washed with PBS and 1 mL of culture medium was added. Forty-eight hours after transfection, the cells were detached with a non-enzymatic cell dissociation buffer (10 min incubation at 37 °C). After centrifugation (7 min, 300 g), the cell pellet was resuspended in staining buffer (PBS supplemented with 5% FBS). High-affinity Fc receptors were blocked by incubation with purified anti-mouse CD16/CD32 (BD Biosciences, Erembodegem, Belgium) for 15 min at 4 °C. Subsequently, the cells were incubated with PerCP-Cy® 5.5 rat anti-mouse CD45 (BD Biosciences) diluted in staining buffer and put on a rotary shaker for 45 min at room temperature for incubation. Following three washing steps with 1 mL staining buffer, the cell pellet was resuspended in 500 μL flow buffer and placed on ice until flow cytometry analysis. Subsequently cells were analyzed using a FACSCaliburTM flow cytometer (BD Biosciences, Erembodegem, Belgium). The fluorescence for the Cy® 5.5-label was measured at 488/690 nm. Data analysis was performed using the FlowJo™ analysis software (Treestar, Costa Mesa, USA).
5
Flow Cytometry Analysis of ASC Surface Antigens
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To confirm no difference between surface antigens expressed on the cellular membrane of ASCs and nano-lantern ASCs, flowcytometric analysis was carried out.
At first, Fc block treatment was performed before antibody staining. The cell suspension was then incubated for 30 min at 4 °C with PE hamster anti-mouse CD29, Alexa Fluor 647 conjugated rat anti-mouse CD34, BV480 rat anti-mouse CD44, PerCP-Cy5.5 rat anti-mouse CD45, PerCP-Cy5.5 rat anti-mouse 7AAD, APC-Cy7 mouse anti-rat CD90/mouse CD90.1, BV421 rat anti-mouse CD105, and PE-Cy7 rat anti-mouse Ly-6A/E (Sca-1) (all from BD Bioscience). After washing the cells twice with PBS, they were analyzed by FACS Canto II (BD Biosciences). The data obtained were analyzed by FlowJo™ software (BD Biosciences).
At first, Fc block treatment was performed before antibody staining. The cell suspension was then incubated for 30 min at 4 °C with PE hamster anti-mouse CD29, Alexa Fluor 647 conjugated rat anti-mouse CD34, BV480 rat anti-mouse CD44, PerCP-Cy5.5 rat anti-mouse CD45, PerCP-Cy5.5 rat anti-mouse 7AAD, APC-Cy7 mouse anti-rat CD90/mouse CD90.1, BV421 rat anti-mouse CD105, and PE-Cy7 rat anti-mouse Ly-6A/E (Sca-1) (all from BD Bioscience). After washing the cells twice with PBS, they were analyzed by FACS Canto II (BD Biosciences). The data obtained were analyzed by FlowJo™ software (BD Biosciences).
6
Murine Lung Cell Profiling by Flow Cytometry
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Fresh lung tissue was used for flow cytometry. The fresh lungs of mice were cut and digested to prepare single lung cells, which were collected after centrifugation and filtered by 40 µm cell strainer. The cell proportion was analyzed by flow cytometry using PerCP-Cy™5.5 rat anti-mouse CD45 (561869, BD Biosciences, 1:100), PE/Cy7 anti-mouse CD31 Antibody (102418, Biolegend, 1:100), BV421 rat anti-mouse CD326 (563214, BD Biosciences, 1:100), PE anti-mouse Pdgfra (12-1401-81, Thermo Fisher, 1:100) or APC anti-mouse Pear1 (labeled APC in our lab with the labeling kit from Thermo Fisher #A20186). The expression of human PEAR1 on humanized mouse primary fibroblast was measured by flow cytometry using APC anti-human PEAR1 (FAB4527R, R&D Systems, 1:100). All the flow cytometry experiments were carried out on aria III, a public technology platform of Shanghai Jiaotong University. The flow cytometry data were analyzed using FlowJo software. The gating strategy of flow cytometry was provided in Supplementary Fig. 14 .
7
Isolation and Analysis of Mouse Microglia
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CX3CR1+/GFP and CX3CR1+/GFP; Sfrp1−/− 10‐ to 12 week‐‐old male littermates were treated with LPS or saline as described. After 3 days, animals were perfused with ice‐cold saline and brains collected on ice‐cold, HBSS Ca2+, Mg2+‐free (Invitrogen). After meninges' removal, cortices were isolated, finely chopped and digested for 20 min in DMEM with GlutaMAX (Invitrogen) containing papain 20 U/ml (Worthington Biochemical Corporation), DNase (50 U/ml, Sigma‐Aldrich) and L‐cysteine (1 mM, MERCK). After addition of 20% FCS (Invitrogen), the tissue was mechanically dissociated and filtered through a 35‐µm nylon strainer (Falcon). Microglial cells were separated from myelin/debris by isotonic Percoll gradient centrifugation (35% Fisher Scientific) at 1,200 g for 45 min at 4°C. The pellet was recovered and sequentially incubated with anti‐mouse CD16/CD32 (1:250, BD Pharmingen; for 15 min at 4°C) followed by rat anti‐CD11b and PerCP‐Cy5.5 rat anti‐mouse CD45 (1:200, BD Pharmingen; for 30 min at 4°C), all in 2% BSA, 5mM EDTA in PBS. After washing, cells were sorted using a BD FACSAria Fusion Flow Cytometer and their signal, size and complexity acquired with DiVA8 Software (BD Pharmingen). Analysis was performed using FlowJo v10.0.7 Software (BD Pharmingen).
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CX3CR1+/GFP and CX3CR1+/GFP; Sfrp1−/− 10‐ to 12 week‐‐old male littermates were treated with LPS or saline as described. After 3 days, animals were perfused with ice‐cold saline and brains collected on ice‐cold, HBSS Ca2+, Mg2+‐free (Invitrogen). After meninges' removal, cortices were isolated, finely chopped and digested for 20 min in DMEM with GlutaMAX (Invitrogen) containing papain 20 U/ml (Worthington Biochemical Corporation), DNase (50 U/ml, Sigma‐Aldrich) and L‐cysteine (1 mM, MERCK). After addition of 20% FCS (Invitrogen), the tissue was mechanically dissociated and filtered through a 35‐µm nylon strainer (Falcon). Microglial cells were separated from myelin/debris by isotonic Percoll gradient centrifugation (35% Fisher Scientific) at 1,200 g for 45 min at 4°C. The pellet was recovered and sequentially incubated with anti‐mouse CD16/CD32 (1:250, BD Pharmingen; for 15 min at 4°C) followed by rat anti‐CD11b and PerCP‐Cy5.5 rat anti‐mouse CD45 (1:200, BD Pharmingen; for 30 min at 4°C), all in 2% BSA, 5mM EDTA in PBS. After washing, cells were sorted using a BD FACSAria Fusion Flow Cytometer and their signal, size and complexity acquired with DiVA8 Software (BD Pharmingen). Analysis was performed using FlowJo v10.0.7 Software (BD Pharmingen).
8
Lung Single Cell Sorting for FACS
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