Fluorescent counting beads

Fluorophore-conjugated antibodies against mouse CD45, CD3, NK1.1, NKp46, DX5, KLRG1, and CD27 were purchased from BD Biosciences, and anti-mouse IL-22 was purchased from Biolegend. Blue-fluorescent reactive dye for distinguishing live and dead cells was purchased from Invitrogen, and dead cells were excluded from all analyses. NK cells were identified by their scatter profile, and as having the surface phenotype CD45⁺CD3⁻NK1.1⁺ among cells from C57BL/6 wild type and Rag2^−/− mice and CD45⁺CD3⁻DX5⁺ among cells from BALB/c Il22^−/− mice. Absolute numbers of cells per sample were calculated by adding a known number of fluorescent counting beads (Spherotech) to each of the samples before analysis on the flow cytometer. Intracellular staining for IL-22 and IFN-γ in isolated lung cells was done after activating cells for 4 hours with Leukocyte Activation Cocktail with Golgiplug (BD Biosciences). Cells were fixed and permeabilized using the Cytofix/Cytoperm Plus kit according to the manufacturer's instructions (BD Bioscience). All samples were analyzed on an LSR II System flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (version 8.2, Tree Star).

All flow cytometric analysis was conducted on BD FACS Aria III or LSR II. The data were analyzed using Flowjo software. To stain for ASCs, CD138 APC or PerCP-Cy5.5 (clone 281-2, BD Biosciences or Biolegend), B220 APC-Cy7 (clone RA3-6B2, Biolegend), CD98 PerCP-Cy5.5 or PE (clone 4F2, Biolegend), CD93 APC (clone AA4.1, Biolegend), CD3 Efluor 450 (17A2, eBioscience), and CD184 BV421 or PE (clone 2B11/CXCR4; BD Biosciences) were used. For B-cell lymphoma 2 (BCL-2 PeCy7; clone BCL/10C4; Biolegend) and mantle cell leukemia (MCL-1 APC; clone D2W9E; Cell Signaling Technologies) staining, cytofix/cytoperm kit (BD) was used. CD45.1 PE (clone A20; Invitrogen) and CD45.2 BV450 (clone 104; BD Biosciences) antibodies were used to distinguish donor and recipient cells in chimera studies. Fluorescent counting beads (Spherotech) were used to quantify cell numbers routinely.