Cell sorting was performed to purify lung epithelial cells and fibroblasts for lung alveolosphere formation assays and to purify lung epithelial cells for transcriptome analysis. For epithelial cell sorting, single-cell suspensions of lung cells were first stained with anti-mouse CD31, CD45, Ter119, CD146, and Epcam antibodies (see Supplementary Table S5 for details). Cells were next stained with streptavidin-APC, followed by incubation with anti-APC microbeads (Miltenyi). Labeled cells were magnetically depleted. Finally, lineage (CD31, CD45, Ter119, and CD146)− propidium iodide− Epcam+ live epithelial cells were sorted using a MoFlo Astrios flow cytometer (Beckman Coulter). For fibroblast cell sorting, single-cell suspensions of lung cells were first stained with anti-mouse CD31, CD45, Ter119, Epcam, and Pdgfra antibodies (see Supplementary Table S5 for details). Cells were stained with streptavidin-APC, followed by anti-APC microbeads (Miltenyi). Lineage (CD31, CD45, Ter119)− propidium iodide− Pdgfra+ live fibroblasts were sorted after magnetic depletion.
Anti apc microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
Anti-APC microbeads are an immunomagnetic cell separation product from Miltenyi Biotec. The microbeads are coated with antibodies specific to the APC (Allophycocyanin) fluorescent dye, allowing for the magnetic separation and isolation of APC-labeled cells.
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Lab products found in correlation
Ls column, by Miltenyi Biotec (11 mentions)
Automacs pro separator, by Miltenyi Biotec (7 mentions)
Facsaria, by BD (7 mentions)
Anti pe microbeads, by Miltenyi Biotec (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Facscanto, by BD (4 mentions)
Dnase 1, by Roche (4 mentions)
Macs column, by Miltenyi Biotec (4 mentions)
Macs microbead technology, by Miltenyi Biotec (3 mentions)
Anti cd45 nanobeads, by BioLegend (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Carl Roth (3 mentions)
Dispase, by Roche (3 mentions)
Collagenase p, by Roche (3 mentions)
Automacs pro, by Miltenyi Biotec (3 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Epcam pe, by BD (2 mentions)
Clone 877621, by R&D Systems (2 mentions)
Mouse igg1 apc, by R&D Systems (2 mentions)
Anti mouse igm microbeads, by Miltenyi Biotec (2 mentions)
112 protocols using anti apc microbeads
1
Purification of Lung Cell Populations
2
Zymosan-Induced Peritoneal Macrophage Isolation
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In addition, we observed the above phenomena using confocal microscopy (TiEA1R, NIKON INSTECH Co. Ltd., Tokyo Japan) under the conditions of with or without AIM coating. Debris was stained with FVD520 (eBioscience). The peritoneal cells derived from the zymosan model mice were stained with APC anti-mouse F4/80 antibody (BioLegend, San Diego, CA) and incubated with Anti-APC microbeads (Miltenyi Biotec). F4/80 positive cells were separated using a MACS LS column (Miltenyi Biotec) stained with CellTracker™ Red CMPTX Dye (10 µM) (Thermo Fisher Scientific).
3
Differentiation of iPSCs to iMSC2 Cells
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The following protocol was used to derive the iMSC2 cell lines from iPSC-DS (iMSC2-DS) and iPSC-WT (iMSC2-WT). Initially proposed by Vodyanik et al. [17 (link),18 (link)], protocol 2 is based on a novel method of iPSC mesenchymal differentiation that requires isolation of a multipotential progenitor at the mesenchymoangioblast stage. For mesendodermal induction, iPSCs at passage 30 were cocultured with OP9 stromal cells. Primitive streak/mesendoderm precursors, which express apelin receptor (APLNR+) [19 (link)], were isolated via MACS sorting on day 2 of OP9 coculture. APLNR-APC antibodies and Anti-APC MicroBeads were used (Miltenyi Biotec).
The isolated APLNR+ progenitors were plated as single cells in semisolid colony-forming serum-free medium (CF-SFM) containing 40% ES-Cult M3120 methylcellulose, 25% serum-free expansion medium (SFEM; Stem Cell Technology), 10% BIT 9500 supplement, and other additives. After 2 weeks, the mesenchymal colony-forming units (MS-CFU) were manually picked, and the iMSC2 cells were transferred to an adherent culture and maintained in EGM-2 medium (Lonza).
The isolated APLNR+ progenitors were plated as single cells in semisolid colony-forming serum-free medium (CF-SFM) containing 40% ES-Cult M3120 methylcellulose, 25% serum-free expansion medium (SFEM; Stem Cell Technology), 10% BIT 9500 supplement, and other additives. After 2 weeks, the mesenchymal colony-forming units (MS-CFU) were manually picked, and the iMSC2 cells were transferred to an adherent culture and maintained in EGM-2 medium (Lonza).
4
Inducible FOXN1 Knockout Mouse Immunization
5
Isolation and Culture of Human Alveolar Cells
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Samples of normal, de-identified human lungs were obtained from non-utilized lungs donated for organ transplantation via an established protocol (PROPEL, approved by University of Pennsylvania Institutional Review Board) with informed consent in accordance with institutional procedures. A 2x2cm piece of distal lung tissue was obtained, pleura and large airways were carefully dissected away, and tissue was processed into a single cell suspension using the same combination of dispase, collagenase I, and DNase used for mouse lungs. A Miltenyi gentleMACS dissociator was used for mincing and incubation for 35min at 37°C. Cells were washed, passed over 70μM and 40μM filters, and RBCs were lysed with ACK lysis buffer. After a single cell suspension was obtained, cells were analyzed by FACS or sorted using the MACS multisort kit, MACs LS columns, and the following antibodies: EPCAM-PE (BD, mouse, Clone 1B7, 1:50), HT2-280 (mouse IgM, a gift of Leland Dobbs, UCSF, 1:50), TM4SF1-APC (mouse, R&D Systems, Clone 877621, 1:100), Mouse IgG1-APC isotype control (R&D systems, 1C002A, 1:100), anti-APC microbeads (Miltenyi, 130-090-855, 1:20), anti-mouse IgM microbeads (Miltenyi, 130-047-302, 1:20). The full protocol for digestion and sorting of human lung epithelial cells, and their propogation as alveolar organoids, has been made available via the Nature Protocol Exchange36 (link).
6
Isolation and Purification of Mouse Hematopoietic Stem Cells
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Bone marrow cells were obtained from the tibia and femur of mice. Lineage-positive (Lin+) cells were depleted by magnetic-activated cell sorting using an APC-conjugated mouse lineage antibody cocktail (BD Pharmingen, San Diego, CA, USA) and anti-APC microbeads (Miltenyi Biotec, Auburn, CA, USA). After magnetic-activated cell sorting purification, the purity of Lin− cells was >95% in all experiments. For in vivo and in vitro donor cell tracking experiments, Lin− cells were labeled with PKH26 (Sigma-Aldrich, St Louis, MO, USA) or Vybrant DiI (Molecular Probes, Eugene, OR, USA) and stained with anti-Sca1 and anti-c-Kit antibodies (BD Pharmingen) and sorted using BD FACSAriaIII (BD Bioscience, San Jose, CA, USA). The purity of each sorted population was >99%.
7
Isolation and Culture of Murine Cardiac Fibroblasts
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As previously described,10 (link) hearts from three 8‐ to 10‐week‐old C57Bl6 mice (Jackson Laboratories) were dissected to isolate ventricular tissue, which was then minced and incubated with 10 mL of digestion solution (10 mg/mL collagenase II, 2.5 U/mL dispase II, 1 μg/mL DNase I, and 2.5 mmol/L CaCl2) for 20 minutes at 37°C. A filtered myocyte‐free single‐cell suspension in PBS containing 0.5% BSA and 2 mmol/L EDTA (PBS/BSA/EDTA) was treated with mouse BD Fc Block (clone 2.4G; BD Biosciences), and immune cells were magnetically removed with CD45 microbeads (Miltenyi Biotec Inc). CD45‐depleted cell suspension was incubation with phycoerythrin (PE)‐conjugated CD31 (clone 390; eBioscience) and allophycocyanin (APC) ‐conjugated CD105 (clone MJ7/19; Biolegend) antibodies. CD31+ endothelial cells were removed using anti‐PE microbeads (Miltenyi Biotec) and CD105+ cardiac fibroblasts were magnetically isolated with anti‐APC microbeads (Miltenyi Biotec) from the flow‐through CD31−‐negative cells. Primary cardiac fibroblasts (CD105+CD31−CD45−) were plated at a density of 2×107 cells/mm2 and cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L glutamine under a humidified atmosphere of air/CO2 (19:1) at 37°C.
8
Isolation and Expansion of Murine ILC2s
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ILC2s were sorted by FACS from the lungs of day 5 N. brasiliensis–infected mice. ILC2s identified as lineage-negative, CD45+ST2+KLRG1+IL7Rα+Sca-1+ cells were either used directly in T cell co-cultures or subsequently expanded in cRPMI (RPMI 1640 [Gibco] supplemented with penicillin/streptavidin [Gibco], l -glutamine [Gibco], and 10% fetal bovine serum [Gibco]) containing 10 ng/ml IL-2 (R&D Systems), 10 ng/ml IL-7 (R&D Systems), and 10 ng/ml IL-33 (R&D Systems) for 5 d.
For in vitro activation studies, lineage-negative cells were sorted by magnetic-activated cell sorting (MACS). Cells were labeled with lineage-APC antibodies and anti–APC-microbeads (Miltenyi Biotec) in MACS buffer (PBS, 2 mM EDTA, and BSA [all Sigma-Aldrich]) according to the manufacturer’s instructions and separated using an AutoMACS system (Miltenyi Biotec). Cells were then cultured in the presence of cytokines for 7 d to generate >95% pure ILC2 cultures. T cells (>95% purity) were isolated from the spleens of naive mice using the mouse CD4 T cell isolation kit according to the manufacturer’s instructions. For OT-2 co-cultures, T cells were isolated from Rag2+/−OT-2tg/+ mice, stained with CellTrace Violet (Molecular Probes) according to the manufacturer’s protocol, and incubated with ILC2s and OVA protein (10 µg/ml) for 5 d.
For in vitro activation studies, lineage-negative cells were sorted by magnetic-activated cell sorting (MACS). Cells were labeled with lineage-APC antibodies and anti–APC-microbeads (Miltenyi Biotec) in MACS buffer (PBS, 2 mM EDTA, and BSA [all Sigma-Aldrich]) according to the manufacturer’s instructions and separated using an AutoMACS system (Miltenyi Biotec). Cells were then cultured in the presence of cytokines for 7 d to generate >95% pure ILC2 cultures. T cells (>95% purity) were isolated from the spleens of naive mice using the mouse CD4 T cell isolation kit according to the manufacturer’s instructions. For OT-2 co-cultures, T cells were isolated from Rag2+/−OT-2tg/+ mice, stained with CellTrace Violet (Molecular Probes) according to the manufacturer’s protocol, and incubated with ILC2s and OVA protein (10 µg/ml) for 5 d.
9
Enrichment of Ag-specific CD8+ T Cells
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Enrichment of Ag-specific CD8+ T cells was performed using an established protocol22 (link). Combined spleen and lymph nodes were digested with collagenase D. Cells were then labeled with APC-conjugated H-2Kd-CSP280–288 tetramer (SYVPSAEQI, NIH Tetramer Core Facility) for 30 min at room temperature, followed by magnetic enrichment over LS columns using anti-APC microbeads (Miltenyi biotec). Enriched samples and unbound fractions were stained with surface antibodies, and Polybead polystyrene microspheres (Polyscience) were used for calculating cell number.
10
Isolation and Cryopreservation of T Cell-Depleted HSCs
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We prepared the HSCs as previously described38 (link). Briefly, we collected BM from sex and MHC-matched MCMs (Table 1 ) by aspirating up to 5 mL from four sites, 20 mL total, and removed red blood cells using ACK lysis buffer (Thermo Fisher Scientific). Next, we washed the cells twice with phosphate-buffered saline and incubated them with anti-TCRα/β allophycocyanin (APC) antibodies (clone R73, BioLegend) for 20 min, followed by incubating with anti-APC microbeads (Miltenyi Biotec) for an additional 20 min at 4 °C. Finally, we passed the stained cells through two LS columns (Miltenyi Biotec) stacked on top of one another, collecting both negative and positive cell fractions, and cryopreserving them at 30 million/mL in serum-free expansion medium (SFEM; Stem Cell Technologies) containing 5% fetal bovine serum and 10% dimethylsulfoxide (DMSO) until further use.
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