To harvest naive BMSCs, we isolated mononuclear cells that express the surface antigen CD271 but not CD45 from bone marrow (Churchman et al., 2012 (link)). In brief, after bone marrow-derived mononuclear cells were collected using the method described above, the cell was labeled with APC-conjugated anti-CD271 (catalog #560326, BD Biosciences) and PE-conjugated anti-CD45 (catalog #555483, BD Biosciences) antibodies in FACS buffer composed of PBS, 1% BSA, and 5 mM EDTA. The fluorescence minus one control was used to set the gates. After washing with FACS buffer three times to remove unbound antibodies and analysis by a cell sorter (BD Biosciences), approximately 10,000 of the CD271+/CD45− cells were recovered. Total RNA was extracted directly from the uncultured CD271+/CD45− cells, and transcriptional profiles of these cells were analyzed using human MSC PCR arrays.
Pe conjugated anti cd45
Manufactured by BD
Sourced in United States
PE-conjugated anti-CD45 is a flow cytometry reagent that binds to the CD45 surface protein expressed on human leukocytes. The PE (Phycoerythrin) fluorescent label allows for the detection and quantification of CD45-positive cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Facsverse flow cytometer, by BD (2 mentions)
Facscalibur, by BD (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Pe conjugated anti cd90, by BD (1 mentions)
Pe conjugated anti cd44, by BD (1 mentions)
Streptavidin allophycocyanin, by BD (1 mentions)
Pe conjugated anti cd31, by BD (1 mentions)
Moflo flow cytometer, by Beckman Coulter (1 mentions)
Facs vantage se diva cell sorter, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Percoll gradient, by GE Healthcare (1 mentions)
Anti fc receptor antibody, by BD (1 mentions)
Papain based neural tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Hamster igg, by Jackson ImmunoResearch (1 mentions)
Cellquest v3, by BD (1 mentions)
70 m cell strainer, by BD (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dnase 1, by Qiagen (1 mentions)
8 protocols using pe conjugated anti cd45
1
Isolation and Transcriptional Profiling of Naïve BMSCs
2
Characterizing Adipose-Derived Stem Cells
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ADSCs were isolated from paratesticular fat of all animals and cultured as described previously.23 (link) Surface markers were identified by flow cytometric analysis of passage 3 ADSCs. Briefly, 1 × 105 ADSCs were harvested and suspended in 500 μl PBS and incubated with phycoerythrin (PE)-conjugated anti-CD90 (BD Bioscience, Sparks, MD, USA), fluorescein isothiocyanate-conjugated anti-CD34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PE-conjugated anti-CD45 (BD Bioscience), or PE-conjugated anti-CD44 (BD Bioscience) antibodies for 15 min at room temperature while protected from light. After washing with PBS 3 times, ADSCs were analyzed using an FACSCalibur (BD Bioscience). Data were analyzed with CellQuest software (BD Biosciences). ADSCs incubated with isotype-matched fluorescent dye-conjugated IgG were used as negative controls.
For cell tracking, ADSCs were labeled with 10 μmol l−1 EdU (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
For cell tracking, ADSCs were labeled with 10 μmol l−1 EdU (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
3
Satellite Cell Isolation from Skeletal Muscle
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Skeletal muscles from both fore‐limbs and hind limbs were dissected out and digested with 0.2% collagenase type II (Worthington Biochemical CorporaAon, Lakewood, Washington, www.worthington-biochem.com ) for 1 hour at 37°C. Then, the digested tissue was filtered through 100 µm‐ and 40 µm‐cell strainers (BD Biosciences). The filtered mononuclear cells were stained with phycoerythrin (PE)‐conjugated anti‐CD31 (BD Biosciences), PE‐conjugated anti‐CD45 (BD Biosciences), FITC‐conjugated anti‐Sca‐1 (BD Biosciences), and biotinylated SM/C‐2.6 antibodies 24 on ice for 30 minutes. After washing, streptavidin‐allophycocyanin (BD Biosciences) was added to the cells labeled with biotinylated SM/C‐2.6 antibody and incubated on ice for 30 minutes. All the cells were resuspended in HBSS (−) and propidium iodide. Cell sorting was performed using MoFlo flow cytometer (BeckMan, Brea, California, www.beckmancoulter.com ), and CD31−, CD45−, Sca‐1−, and SM/C‐2.6+ cells were collected as satellite cells 24 . Percentage of satellite cells in the total mononuclear cells, except for CD31‐positive endothelial cells and CD45‐positive lymphocytes/leukocytes, was calculated for analyzing satellite cell population.
4
Isolation and Characterization of Astrocytes and Microglia
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Brains and spinal cords from transgenic GFAP-EGFP mice were dissociated using the papain-based neural tissue dissociation kit (Miltenyi Biotec, Germany). Myelin was separated from the cells on a discontinuous Percoll gradient (GE Healthcare Biosciences AB, Uppsala, Sweden) and the cells were washed and incubated with blocking solution containing HBSS, FBS (Sigma-Aldrich), anti-Fc receptor antibody (BD Biosciences, Brøndby, Denmark), hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), and sodium azide. The cells were then labeled with phycoerythrin (PE)-conjugated anti-CD45 (BD Biosciences) for 15 min at 4 °C and propidium iodide (PI, Sigma-Aldrich) to detect microglia/macrophages and non-viable cells, respectively.
Cells were sorted using a FACSVantage SE DiVa cell sorter (BD Biosciences). Astrocytes were defined as EGFP positive and CD45 negative (Fig.6 b). Microglia were defined as EGFP negative and CD45dim. Sorted astrocytes and microglia were re-analyzed by flow cytometry and quantitative real-time RT-PCR to verify purity.
Cells were sorted using a FACSVantage SE DiVa cell sorter (BD Biosciences). Astrocytes were defined as EGFP positive and CD45 negative (Fig.
5
Flow Cytometric Analysis of Hematopoietic Cells
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Adherent and floating cells were collected and filtered through a 70-µm cell strainer (BD Biosciences). The cells were then washed twice with FACS buffer (PBS, 10 mM EDTA, and 2% of fetal bovine serum) and resuspended in Dulbecco's PBS at up to 5×106 cells per 100 µL. The cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD43 antibody, phycoerythrin (PE)-conjugated anti-CD45, peridinin chlorophyll protein (PerCP)-conjugated anti-CD34, and FITC-conjugated anti-KDR antibodies (BD Biosciences) for 30 min on ice, washed, and resuspended in staining buffer. The samples were analyzed using a FACSVerse flow cytometer (BD Biosciences) with the CellQuest v3.3 software (BD Biosciences). Data were collected and analyzed in the Flowjo software (Flowjo, LLC, Ashland, OR, USA).
6
Isolation and Characterization of Intestinal Stem Cells
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Small intestines were dissected from 2 months old Lgr5EGFP-Cre-ERT males, cut in pieces of 2 mm2, placed into 50 ml conical tubes and washed three times for 10 min with 40 ml of PBS/5 mM EDTA on a rocking plate at room temperature. Villi were mechanically removed by gentle shaking three times and trituration using a 10 ml pipette. Villi material was collected by centrifugation at 200g for 3 min and washed twice with PBS. Crypts were separated from the intestine by vigorous shaking two times passing through a 70 μM filter (BD Falcon). Crypt material was collected by centrifugation at 200g for 3 min, washed twice with PBS and incubated for 5–10 min with 0.15 mg/ml collagenase (Sigma) and 0.1 mg/ml DNase I (Qiagen) at 37°C on a rocking plate. Single cell suspensions were washed twice and resuspended in PBS supplemented with 2% fetal calf serum. Cells were stained with PerCP-eFluor® 710-conjugated anti-EpCAM 1:400 (eBioscience), PE-conjugated anti-CD45 at 1:400 (BD Biosciences) and anti-CD31 at 1:400 (BD Biosciences) antibody for 30 min at room temperature. Living cells were gated by DAPI dye exclusion. Enterocytes were isolated as EpCAM+CD31−CD45−DAPI−. ISCs were isolated as Lgr5-EGFPhighCD31−CD45−DAPI−. Fluorescence-activated cell sorting was performed using BD FACS Aria III SORP cell sorter (85 μM nozzle) and analysed using FlowJo software.
7
Characterizing EPO-Treated Bone Marrow Stem Cells
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BMSCs were trypsinized after incubation with or without EPO (500 IU/ml) for 48 h and washed three times with phosphate-buffered saline (PBS). Cell surface markers were examined by immunostaining with the following antibodies: phycoerythrin (PE)-conjugated anti-CD45 (BD Biosciences, San Jose, CA), fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BD Biosciences), and FITC-conjugated anti-CD90 (BD Biosciences) antibody. The labeled cells were analyzed using a flow cytometer (BD LSRFortessa™, Piscataway, NJ). Osteogenic and adipogenic differentiation potential of the cells in the two groups was examined according to the manufacturer’s protocol (Cyagen, China).
8
Flow Cytometric Analysis of Brain Inflammation
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To measure inflammatory-cell infiltration in the brain, flow cytometry was performed. Single-cell suspensions were prepared from each mouse brain, washed with 2% fetal bovine serum in PBS, and incubated with Fc block™ (BD Biosciences, San Jose, CA, USA) for 10 min at 4 °C. After washing twice with 2% FBS in PBS, the cells were incubated with PE-conjugated anti-CD45, FITC-conjugated anti-CD11b, and APC-conjugated anti-Ly6G antibodies (BD Biosciences) for 30 min at 4 °C. A BD FACSVerse™ flow cytometer (BD Biosciences) was used to measure the microglia (CD45med/CD11b+/Ly6G−), leukocyte (CD45high), macrophage (CD45high/CD11b+/Ly6G−), and neutrophil (CD45high/CD11b+/Ly6G+) populations as defined elsewhere [28 (link), 29 (link)]. The data were collected and analyzed using BD FACSuite™ software (BD Biosciences).
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