Percp cy5.5 cd44 im7

MSU crystals (1 mg suspension/dose) in 50 μl PBS or PBS alone were administered i.n. once/day, daily for 3 days, to Il5^+/venus and Il13^+/eGFP mice or wild-type mice. Twenty-four hours after the last administration, lungs were collected and minced using a gentleMACS Dissociator (Miltenyi Biotec, Auburn, CA), and digested with Liberase^™ Research Grade (Roche, Mannheim, Germany) in RPMI 1640 medium in the presence of DNase I solution (STEMCELL Technologies, Vancouver, Canada) for 1 hr at 37°C. After digestion, single lung cells were hemolyzed with ammonium-chloride-potassium (ACK) buffer, and washed with phosphate buffered saline (PBS) containing 0.1% sodium azide and 1% bovine serum albumin. To examine the expression of cytokines by group 2 innate lymphoid cells (ILC2s), lung single cell suspensions were stained with a PE-conjugated lineage cocktail [CD3 (145-2C11), CD14 (rmC5-3), CD16/32 (2.4G2), B220 (RA3-6B2)], APC-CD25 (PC61), and PerCP-Cy5.5-CD44 (IM7, BD Biosciences). Lung ILC2s were identified as Lin^-CD25⁺CD44^hi cells as previously described (25 (link)). The expression levels of IL-5venus and IL-13eGFP by CD3⁺ T cells or ILC2s were detected by FACS (BD FACSCalibur, BD Biosciences).

Lungs were minced using a gentleMACS Dissociator (Miltenyi Biotec, Auburn, CA), and digested with Liberase^TM Research Grade (Roche, Mannheim, Germany) in RPMI 1640 medium in the presence of DNase I solution (STEMCELL Technologies, Vancouver, Canada) for 1 hour at 37 °C. After digestion, single lung cells were hemolyzed with ammonium-chloride-potassium (ACK) buffer, and washed with PBS containing 0.1% sodium azide and 1% bovine serum albumin (PAB). To quantitate the numbers of lymphocytes, single lung cells were stained with FITC-CD3e (145-2C11), phycoerythrin (PE)-B220 (RA3-6B2), peridinin chlorophyll protein complex (PerCP)-CD4 (RM4-5), and allophycocyanin (APC)-CD8 (53-6.7, BD Bioscience, San Jose, CA) for 30 min at 4°C. After washing in PAB buffer, the expression levels of CD3, CD4, CD8, and B220 were detected by FACS (BD FACSCalibur, BD Bioscience). To examine the expression levels of cytokines by type 2 innate lymphoid cells (ILC2s) or CD4⁺ T cells, single lung cells from Il13^+/eGFP mice or Il5^+/venus mice were stained with a PE-conjugated lineage cocktail [CD3 (145-2C11), CD14 (rmC5-3), CD16/32 (2.4G2), B220 (RA3-6B2)], APC-CD25 (PC61), PerCP-Cy5.5-CD44 (IM7, BD Biosciences) or PE-CD4, and PerCP-CD3. Lung ILC2s and CD4⁺ T cells were identified as Lin⁻CD25⁺CD44^hi cells and CD3⁺CD4⁺ cells, respectively, as previously described (24 (link)).