Protocol detail

Isolation and Purification of Mouse Cardiac Macrophages

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Mouse hearts were isolated and enzymatically digested, as previously described ^{5 (link)}, with the inclusion of 1mM Flavopiridol in the digestion buffer. Digestions were stopped after 20min and cells were processed into a single cell suspension on ice. Cells were stained with CD45 magnetic beads (Miltenyi Biotec) and subsequently fluorescently tagged antibodies against CD45, CD64 and CD11b. Hematopoietic cells were positively enriched using the AutoMacs instrument (Miltenyi Biotec) and live macrophages (CD45⁺, CD64⁺, CD11b⁺, DAPI^Neg) were sorted on the Aria Fusion (BD Bioscience) under low pressure into DMEM containing 50% FCS for cell visualization and 10X single cell RNA sequencing or RLT buffer (Qiagen) for RNA extraction.

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Dick S.A., Macklin J.A., Nejat S., Clemente-Casares X., Momen A., Kantores C., Hosseinzadeh S., Barbu I., Chen J., Althagafi M.G., Besla R., Wong A., Aronoff L., Zaman R., Lavine K.J., Razani B., Ginhoux F., Husain M., Cybulsky M.I., Robbins C.S, & Epelman S. (2018). Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nature immunology, 20(1), 29-39.

Publication 2018

CD45 magnetic beads AutoMacs instrument Aria Fusion RLT buffer

Antibodies Aria Buffer Cd11b Cell Digestions Flavopiridol Hearts Hematopoietic Macrophages Mouse Pressure

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Variable analysis

independent variables

Inclusion of 1mM Flavopiridol in the digestion buffer

dependent variables

Live macrophages (CD45+, CD64+, CD11b+, DAPIneg)

control variables

Mouse hearts were isolated and enzymatically digested, as previously described [5]
Cells were stained with CD45 magnetic beads (Miltenyi Biotec) and subsequently fluorescently tagged antibodies against CD45, CD64 and CD11b
Hematopoietic cells were positively enriched using the AutoMacs instrument (Miltenyi Biotec)
Live macrophages were sorted on the Aria Fusion (BD Bioscience) under low pressure into DMEM containing 50% FCS for cell visualization and 10X single cell RNA sequencing or RLT buffer (Qiagen) for RNA extraction

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