Anti mouse cd45 antibody

Lungs were perfused with cold PBS via right ventricle, minced and digested with collagenase IV (5 mg/ml) and DNase I [24 (link)]. Lung digests were passed through 70 μ filter, cells were harvested by centrifugation, and treated with RBC lysis buffer. Cells were then washed and suspended in PBS.
To determine differential cell counts, an aliquot of cell suspension equivalent to 1 x 10⁶ cells was labeled with antimouse CD45 antibody conjugated with magnetic microbeads (Miltenyi Biotec Inc, Auburn, CA) to isolate CD45 positive cells. Cytospins of CD45 positive cells were prepared, stained with DiffQucik and number of macrophages, neutrophils and T cells were determined.
For flow cytometry analysis, cells obtained after RBC lysis were incubated with Zombie UV^™ (BioLegend) to label dead cells and stained with fluorescence-labeled Abs against surface markers of leukocytes, such as CD45, CD11c, CD11b, F4/80, CD3e, CD8 and CD4. Appropriate isotype-matched controls and fluorescence minus one (FMO) were used in all experiments. All antibodies were purchased from BioLegend (San Diego, CA). Cells were fixed and analyzed in BD LSR II Flow cytometerI (BD Biosciences) and data was analyzed using FlowJO version 10 (Tree Star, Ashland, OR).

Cells were isolated from the bone marrow, spleen, lymph nodes, and lung as previously described [11 , 22 , 23 (link)]. After hypotonic lysis to remove erythrocytes, single cell suspensions were prepared. CD45^negative (non-hematopoietic cells) were isolated from the lung. To do so, 1% low melting point agarose was inserted into the lung via the trachea, and the tissue was then digested with dispase. CD45^negative cells were isolated from the resulting cell suspension using a magnetic enrichment beads conjugated to an anti-mouse CD45 antibody (Miltenyi Biotec, Auburn, CA). Genomic DNA was isolated from cell suspensions using a QIAamp DNA Mini Kit (QIAGEN), and PCR to detect the excised Ahr allele was performed as previously described [20 (link), 23 (link), 24 (link)].

CSCs were isolated from adult transgenic and wt hearts by enzymatic methods.^{35 (link), 40 (link)} For CD45⁻c-Kit⁺ cell purification, myocyte-depleted small cardiac cells were incubated with microbeads conjugated with anti-mouse CD45 antibody (Miltenyi Biotec S.r.l., Calderara di Reno (BO), Italy) and removed from the preparation by magnetic-activated cell sorting (Miltenyi). From the CD45⁻ fraction, the c-Kit⁺ (CD45⁻) cardiac cells were enriched through incubation with a microbeads-conjugated mouse monoclonal antibody against c-Kit (Miltenyi) and separated by magnetic-activated cell sorting.
Freshly isolated CD45⁻c-Kit⁺ cardiac cells were cultured on gelatin-coated dishes in CSC growth medium^{35 (link), 40 (link)} before clonogenic, spherogenic and BrdU assays.
Cardiosphere myogenic differentiation was performed as previously described.^{35 (link)}Endothelial differentiation was obtained by culturing the CSC for 3–10 days in MEM Alpha (Life Technologies), 10% ESQ-FBS (Life Technologies), 1 μM dexamethasone, 50 μM/ml ascorbic acid, 10 mM β-glycerophosphate (all from Sigma-Aldrich, Milano, Italy) and 10 ng/ml VEGF (PeproTech EC Ltd., London, UK). LY294002 (10 μM, Calbiochem, San Diego, CA, USA) and UO126 (10 μM, Cell Signalling Technology, Danvers, MA, USA) were added to the culture for AKT and ERK1/2 inhibition.