Cells from adipose tissues were prepared and lysed with ACK lysing buffer (Gibco) to exclude red blood cells and were stained with antibodies on ice for 30 min in the dark. APC/Cy7 anti-mouse CD45 (30-F11, BioLegend), Percpcy5.5 anti-mouse CD11b (M1/70, eBioscience), and APC anti-mouse F4/80 (BM8, eBioscience) were used to stain ATMs, and BV605 anti-mouse Mer (108928, BD Biosciences), PE anti-mouse CD64 (X54-5/7.1 BioLegend), FITC anti-mouse CD11c (HL3, BD Biosciences), PE/Cy7 anti-mouse CX3CR1 (SA011F11, BioLegend), and Alexa Fluor 700 anti-mouse Ly6C (HK1.4, BioLegend) were used to further analyze surface markers of ATMs, BV421 anti-mouse CD45.1 (A20, BioLegend) and PE/Cy7 anti-mouse CD45.2 (104, eBioscience) were used to identify ATMs from CD45.1 and CD45.2 mice, and all antibodies were used in 1:400 dilutions except CD64 (used in 1:200 dilutions). Cells were washed three times and they were performed on FACSFortessa or FACSAria III flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
Anti mouse cd45 pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse CD45-PE-Cy7 is a fluorescently labeled antibody that binds to the CD45 antigen expressed on the surface of mouse cells. It is a flow cytometry reagent used for the identification and enumeration of mouse leukocyte populations.
Automatically generated - may contain errors
Lab products found in correlation
Facs aria 3 machine, by BD (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti mouse epcam pe, by Thermo Fisher Scientific (2 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
Percpcy5.5 anti mouse cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse f4 80 bm8, by Thermo Fisher Scientific (1 mentions)
Fitc anti mouse cd11c hl3, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Apc cy7 anti mouse cd45, by BioLegend (1 mentions)
Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Fc receptor blocking antibody, by R&D Systems (1 mentions)
Flow cytometry antibodies, by BioLegend (1 mentions)
Moflo astrios eq, by Beckman Coulter (1 mentions)
Flow cytometry staining buffer, by Thermo Fisher Scientific (1 mentions)
Isoflow, by Beckman Coulter (1 mentions)
Tri reagent, by Zymo Research (1 mentions)
Facsaria 2, by BD (1 mentions)
7 protocols using anti mouse cd45 pe cy7
1
Isolation and Analysis of Adipose Tissue Macrophages
2
Measuring Macrophage Viability and Apoptosis
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For cell viability, BMDMs were washed with PBS 72 h after siRNA transfection and collected for counting cell number with trypan blue staining. Cell death was analyzed using an Annexin V Apoptosis Detection Kit (88–8007-72; eBioscience) according to the manufacturer’s instructions. For the adoptive transfer experiments, 18 h after cell transfer, peritoneal cells from CD45.1 mice were collected and stained with APC/Cy7 anti-mouse CD45 (1:400, 103116; BioLegend), Brilliant Violet 421 anti-mouse CD45.1 (1:400, 110732; BioLegend), and PE/Cy7 anti-mouse CD45.2 (1:400, 25–0454-80; eBioscience) for identification of transferred macrophages. Cells were analyzed on an LSR Fortessa flow cytometer (BD Biosciences) using FlowJo software (BD Biosciences).
3
Corneal Cell Phenotyping by Flow Cytometry
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Transplanted corneas were collected and digested with collagenase and Dnase, and single-cell suspensions were prepared and filtered. Cells were incubated with an Fc receptor blocking antibody (R&D Systems, Minneapolis, MN, USA). After they were washed, the cells were incubated with flow cytometry antibodies from BioLegend (San Diego, CA, USA) or Thermo Fisher Scientific for 1 hour: PE-Cy7-anti-mouse CD45; Pacific Blue-anti-mouse CD11b Ab; FITC-anti-mouse CD4 Ab; APC-anti-mouse Ly-6G Ab; and PE-anti-mouse F4/80 Ab. Stained cells were rinsed and analyzed using an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the results were analyzed using Summit 4.3 (Agilent Dako, Santa Clara, CA, USA). Dead cells were excluded by performing forward-scatter versus side-scatter analysis, and samples were subsequently analyzed by gating on CD45+ cells (Supplementary Fig. S1 ).
4
Isolation of Cerebrovascular Endothelial Cells
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Sham and CCI injured tissues were prepared as for flow cytometry at 1 dpi as described above. Cortical cells were incubated for surface staining with PE-Cy7 anti-mouse CD45 (ThermoFischer) 1:100 and BV421 rat anti-mouse CD144 (VE-Cadherin) (BD Horizon) 1:100 pre-conjugated antibodies, for 20 min at 4 °C, diluted in FcR blocking solution. Cells were resuspended in 0.5 mL flow cytometry staining buffer (ThermoFischer) and run on a Beckman Coulter MoFlo Astrios EQ using a 100 μm nozzle at 25 psi at a sort rate of about 10,000 events/second using IsoFlow (Beckman Coulter). Debris were gated out using a Forward Scatter Area x Side Scatter Area plot. Aggregates were excluded using a Forward Scatter Height x Forward Scatter Width and a Sideward Scatter Height x Sideward Scatter Width plot. CD45+ cells were excluded and cvECs were sorted based on BV421 expression using CD45 PE-Cy7 log Area by a CD144 BV421 log Area plot. Post sort purities for CD45-/CD144+ cvEC population was >95%. Cells were collected directly into 250 μL TRI Reagent (Zymo Research, Irvine, CA, USA) for subsequent RNA extraction.
5
Isolation and Analysis of Ascites-Derived Cells
6
Isolation and Analysis of Ascites-Derived Cells
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10-13 week old REM2-KPf/fC mice were anesthetized and approximately 100μl of peripheral blood and ascites was collected in PBS containing 5mM EDTA and 2% Dextran. Samples were incubated at 37°C and red blood cells were lysed using RBC lysis buffer (eBiosciences). Remaining cells were stained with anti-mouse EpCAM-PE (eBiosciences) and anti-mouse CD45-PE-Cy7 (eBiosciences) antibodies. Analysis was carried out on a FACSAria III machine (Becton Dickinson) and data analyzed with FlowJo software (Tree Star).
7
Flow Cytometric Analysis of Cell Markers
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