Pe cy7 anti mouse cd45

Mouse adipose-derived stem cells (ASCs) were isolated from inguinal subcutaneous fat of C57BL/6 mice that were fed normal chow. The cells were cultured as described previously [12 (link)]. Briefly, subcutaneous adipose tissues were digested with collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) in PBS (phosphate-buffered saline) by incubation in a shaker at 37 °C for 30 min. The digestion was terminated by the addition of 10% fetal bovine serum (FBS), and centrifuged at 1200 rpm for 5 min. Cells were suspended in complete medium made of DMEM/F12 (Dulbecco modified Eagle medium) medium supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were cultured in 37 °C at 5% CO₂ incubator. ASCs at passage 3–5 were used in all the experiments.
ASCs were identified by FACS as described previously [12 (link)] with fluorescence-conjugated anti-mouse antibodies, FITC Hamster Anti-Rat CD29 (BD Pharmingen; Cat.555005), Alexa Fluor 647 Rat anti-Mouse CD34 Clone RAM34 (RUO) (BD Pharmingen; Cat.560233), APC Rat Anti-Mouse CD90.2 Clone 53-2.1 (RUO) (BD Pharmingen; Cat.561974), APC anti-mouse CD105 (Biolegend; Cat.120413), PE anti-mouse CD31 (Biolegend; Cat.102407), or PE/Cy7 anti-mouse CD45 (Biolegend; Cat.103113), according to the manufacturer’s instructions. Flow cytometry was conducted on a Becton-Dickinson LSR I analyzer.

C57BL/6J or apoA1 TG mice were administered 100 µg zymosan A (Sigma-Aldrich) in PBS. After 16 hr mice were sacrificed and peritoneal exudates collected by lavage with 5 ml of ice cold sterile PBS with 2 mM EDTA. Total cell counts and cellular composition of peritoneal exudate were determined as previously described (Iqbal et al., 2014 (link)). Antibodies used to identify neutrophils (CD45⁺/CD115^lo/Ly6G⁺) and monocytes (CD45⁺/CD115^hi/Ly6C^hi/lo) were APC anti-mouse Ly-6G/Ly-6C (Gr-1), PE anti-mouse CD115 (CSF-1R), and PE/Cy7 anti-mouse CD45 from Biolegend (San Diego, CA). Total blood cell counts were determined with a Genesis blood counter (Oxford Science, CT).

To quantify neutrophil infiltration, the bronchoalveolar lavage fluid (BALF) from immunized mice 24 h post-challenge was collected and centrifuged. The pellet was stained using the following antibodies: PE/Cy7 anti-mouse CD45 and APC/Cy7 anti-mouse Ly-6G (Biolegend). Then, the samples were analyzed using BD FACSArray software TM on a BD FACS Array flow cytometer (BD Biosciences). In addition, the concentrations of pro-inflammatory cytokines in the supernatant, such as IL-1β and TNF-α, were quantified by a Mouse Quantikine ELISA. The protocol was performed based on the manufacturer's (Biolegend) instructions.