Figure 3.
CD11bhi MΦ, but not Siglec-Fhi MΦ, numbers are decreased in cFLIPΔ/Δ mice after bleomycin. Flow cytometry was used to assess total MΦ numbers 0, 4, 7, and 14 days after bleomycin in lavage and lung digests. cFLIPΔ/Δ and cFLIPfl/fl mice were started on doxycycline 1 day after bleomycin. (A–C) Total MΦ, Siglec-Fhi MΦ, and CD11bhi MΦ numbers in BAL. (D–F) Total MΦ, SiglecF+ MΦ, and CD11bhi MΦ numbers in lung digests. (G) Bleomycin-treated mice were given IT α-CD45 to label alveolar cells (IT CD45). BAL was performed 3 minutes later to sample alveolar cells and remove unbound antibody. Lung digestion was performed immediately after lavage. BAL and lung digest was processed for flow cytometry; Siglec-Fhi MΦ and CD11bhi MΦ were separated as described in Figure 1A. IT CD45 staining is shown in BAL and lung digest. BAL staining acts as a positive control; lung digest from mice not given IT CD45 help set the negative gate. (H) CD11bhi MΦ can be separated into high-and low-SSC populations using SSC versus CD11c. (I) Low-SSC CD11bhi MΦ correlate with IT− tissue MΦ, whereas high-SSC CD11bhi MΦ correlate with IT+ alveolar MΦ. (J) Representative dot plots showing high- and low-SSC subpopulations in cFLIPfl/fl and cFLIPΔ/Δ mice 14 days after bleomycin. (K) Total numbers of high- and low-SSC CD11bhi MΦ 14 days after bleomycin corresponding to alveolar and tissue CD11bhi MΦ, respectively. Data from n = 3–5 mice/group shown as mean (±SEM). *P < 0.05, **P < 0.01.