Dissociated cells were incubated with antibodies (APC anti-α7-Integrin, AbLab #67-0010-05, Vancouver, Canada; APC anti-β1-Integrin, eBioscience #17-0291-82; RRID:AB_1210793, San Diego, CA; PE-Cy7 anti-CD31, BD Biosciences Cat#561410; RRID:AB_10612003, San Jose, CA; Biotin anti-CD34, eBioscience Cat#13-0341-81; RRID:AB_466424; PE-Cy7 anti-CD45, BD Biosciences Cat#552848; RRID:AB_394489; Biotin anti-CXCR4, eBioscience Cat#13-9991-82; RRID:AB_10609202; Biotin anti-VCAM-1, BD Biosciences Cat#553331; RRID:AB_10053328; PE streptavidin, BD Biosciences Cat#554061; all at 0.5 μl per 1 million cells) on ice for 30 min. Propidium iodide (PI) (1 μg/ml, Sigma #P4170) was added to differentiate between live and dead cells. Only live cells (PI−) were counted. FACS analysis and cell sorting were performed in a BD FACSAriaII (BD Biosciences, San Diego, CA) using the FACSDiva software (BD Biosciences). Single-cell precision was used for sorting single cells into 96-well plate for clonal analysis, and 4-way purity precision was used for bulk sort. Data were analyzed using FlowJo (FLOWJO LLC, Ashland, OR). Further information on antibodies used is listed in Key Resources Table .
Pecy7 anti cd45
Manufactured by BD
Sourced in United Kingdom
PECy7 anti-CD45 is a fluorochrome-conjugated antibody that binds to the CD45 antigen expressed on the surface of human leukocytes. It is a tool used in flow cytometry and other immunoassays to identify and quantify CD45-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (4 mentions)
Anti cd11c alexa700, by Thermo Fisher Scientific (4 mentions)
Anti mhcii fitc, by Thermo Fisher Scientific (4 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Liberase tl, by Roche (2 mentions)
Anti cd11b pe, by BD (2 mentions)
Anti f4 80 apc, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Collagenase, by Thermo Fisher Scientific (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Anti cd16 32, by Thermo Fisher Scientific (2 mentions)
Anti cd103 pe, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Anti cd86 pe, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Lps free fbs, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Streptavidin pe, by BD (1 mentions)
Anti cd31 pe cy7, by BD (1 mentions)
10 protocols using pecy7 anti cd45
1
Isolation and Characterization of Single Cells
2
MDSC Depletion in Tumor-Bearing Mice
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When tumors were palpable, mice were injected (i.p.) with anti-Gr1 (clone RB6-8C5, BioXCell) or control antibody (clone LTF-2, BioXCell) (200µg/injection, in 100µl PBS) every 48 hours. MDSC depletion was confirmed in the peripheral blood of recipient mice. Blood sample was recovered in heparin containing tubes. After red blood cell lysis by ACK, nucleated cells were incubated for 20 minutes at 4°C with blocking antibody (24G2, 1mg/ml, 1/100). Cells were stained for 30 minutes at 4°C with the following antibodies: PECy7 anti-CD45 (Clone 30-F11, BD Pharmingen, 1/800), eF450 anti-CD11b (Clone M1/70, eBioscience, 1/100), PerCPCy5.5 anti-Gr1 (clone RB6-8C5, BD Pharmingen, 1/100), PE anti-CD11c (Clone N418, eBioscience, 1/100) and LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit (Life Technologies, L34959, 1/100). Staining was assessed with a FACS Fortessa cytometer, and flow cytometry data were analyzed using FACSDiva (BD Biosciences) or FlowJO (Tree Star) software.
3
Isolation and Phenotyping of CNS Immune Cells
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The mice were anesthetized and then transcardially perfused with cold PBS. The brains were isolated and placed in 2 ml of ice-cold Hank's Balanced Salt Solution (HBSS). To facilitate dissociation, 123 μl of Liberase TL (05401020001, Roche) and 5 μl of 100 mg/ml DNase I (DN25, Sigma) were added to the brain tissues and incubated for 30 min at 37 ℃. After enzymatic dissociation, the cells were resuspended in 30% Percoll (17089101, Citiva, Sweden) and centrifuged for 25 min at 2850 rpm without a brake. Myelin was removed, and the pelleted cells were washed with HBSS. Red blood cells (RBC) were lysed using RBC lysis buffer (420301, BioLegend), and the cells were then counted using AO/PI (CS2-0106, Nexelom Bioscience, MA, USA) staining and a Fluorescent cell counter (Cellometer K2, Nexelom Bioscience, MA, USA).
CNS immune cells were stained with the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (L34962, Invitrogen) and incubated with Fc-Receptors blocker (101302, BioLegend). The cells were then labeled with the following antibodies: PECy7 anti-CD45 (561868, BD), FITC anti-CD11b (101206, BioLegend), PE anti-P2ry12 (848004, BioLegend).
CNS immune cells were stained with the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (L34962, Invitrogen) and incubated with Fc-Receptors blocker (101302, BioLegend). The cells were then labeled with the following antibodies: PECy7 anti-CD45 (561868, BD), FITC anti-CD11b (101206, BioLegend), PE anti-P2ry12 (848004, BioLegend).
4
Isolation and Analysis of Kidney Leukocytes
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Animals were perfused with cold normal saline and contralateral and UUO kidneys were placed on ice, minced, digested with Liberase TL (Roche) with 1% DNase (Sigma-Aldrich), then placed at 37°C for 10 minutes and vortexed intermittently. An equal volume of ice-cold HBSS+10%FBS was added to stop Liberase/DNase. Glomeruli were removed by passing cell suspension through a 40 μm Nylon filter. Cells were stained per protocol with DAPI, PE-Cy7-anti-CD45, PE-anti-CD11b, APC-eFluor780-F4/80 from BD Sciences. Cells were blocked with mouse Fc Block (BD Biosciences). Leukocytes were identified and gated based on their positive F4/80 expression. Data was acquired on the LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc).
5
Isolation of Tumor-Infiltrating Cells
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Mice were anesthetized with ketamine/ xylazine, then tumour tissues or organs were harvested and incubated with PBS containing 1mg/ml collagenase I (Gibco), 2 mg/ ml Dispase I (Gibco), 100ug/ml DNase I (Roche) and 2 mM CaCl2 for 30 min at 37 °C. After incubation, the digested tissue was passed through a cell strainer and then washed by PBS including 2% FBS. For red cell exclusion, we incubated samples with red cell lysis buffer (Sigma) for 5 min at 37 °C and then washed by PBS including 2% FBS and 2 mM EDTA.
Cells were stained with the following monoclonal antibodies: PE/ Cy7 anti-CD45 (552848, BD), APC anti-CD31 (561814, BD). Data acquisition was performed with BD FACSVerse and analysis was performed with BD FACSuite software, and FlowJo software.
Cells were stained with the following monoclonal antibodies: PE/ Cy7 anti-CD45 (552848, BD), APC anti-CD31 (561814, BD). Data acquisition was performed with BD FACSVerse and analysis was performed with BD FACSuite software, and FlowJo software.
6
Colon and Caecum Immune Cell Isolation
7
Immunophenotyping of Mouse Immune Cells
8
Dendritic Cell Isolation and Stimulation
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Bone marrow was harvested and cultured as previously described[23 (link)]. On day six of the culture the cells were harvested from the plates and semi-adherent cells removed. The cells were spun at 400g and re-suspended at a concentration of 1×106 cells/ml in DC medium (RPMI 1640 supplemented with 10% LPS-free FBS (Gibco, Paisley, UK) 1% Penicillin/streptomycin and 50mM Beta-mercaptoethanol (Sigma Aldrich, Dorset UK)). The cells were stimulated overnight with LPS (100ng/ml) or T. muris antigen (5μg/ml). After 24 hours the cells were harvested and prepared for flow cytometry. FcR were blocked by incubating cells in anti-CD16/32 (2μg/ml) for 15 minutes. Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson), anti-CD11c Alexa700 (1μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-CD86 PE (1 μg/ml) (all E Bioscience) and acquired using a LSRII flow cytometer (Becton Dickinson Biosciences, Oxfordshire). Data was analysed using Flow Jo flow cytometry analysis software (Tree Star, Inc, Oregon, US).
9
Colon and Caecum Immune Cell Isolation
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Caecum and colon were harvested at autopsy and digested in RPMI containing 5% L-glutamine, 5% penicillin streptomycin, 10% fetal bovine serum (Sigma Aldrich, Dorset, UK), collagenase (1mg/ml), and dispase (0.5mgs/ml, both Gibco, Paisley, UK) for 2 hours at 37 °C. Cells were then forced through a 70μm cell strainer, centrifuged at 405g for 5 minutes and resuspended in 10mls 80% Percoll (GE Healthcare, Buckinghamshire, UK) solution which was then overlaid on a 40% Percoll solution. Cells were centrifuged for 25 minutes at 1000g and the cells at the gradient interface harvested. Fc receptors were blocked using anti-CD16/32 (2 μg/ml E bioscience, Hatfield, UK). Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson, Oxford, UK), anti-CD103 PE (1 μg/ml), anti-CD11c Alexa700 (2.5 μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-F4/80 APC (1 μg/ml) (all E bioscience, Hatfield, UK) and acquired by flow cytometry on the BD LSRII. Data was analysed using FlowJo flow cytometry software (Tree Star inc. Oregon, US).
10
Dendritic Cell Isolation and Stimulation
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