Anti mouse cd45 fitc

Mouse lungs were minced to single cell suspensions and filtered through 70‐μm cell strainers (BD Biosciences, San Jose, CA) as previously described.9, 28 The erythrocytes were lysed using red blood cell lysis buffer (BD Biosciences) and washed with phosphate‐buffered saline. Cells were labeled with monoclonal antibodies including anti‐mouse CD45‐FITC, Gr‐1‐PE, CD11b‐PerCP, CD48‐APC, and counting beads (BD Biosciences) was added. Matched isotype antibodies were used as negative controls. All samples were analyzed using a FACSCalibur cytometer (BD Biosciences) and CellQuest software, and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

The following group designations are used throughout the manuscript: Rag/PBS and DKO/PBS–PBS-injected Rag2^-/- or Rag2^-/-NHE3^-/- double knockout (DKO) mice, respectively; Rag/AT and DKO/AT—Rag2^-/- or Rag2^-/-NHE3^-/- DKO mice adoptively transferred with 5x10⁵ cells in 500 μl PBS injected intraperitoneally. Naïve T cell transfer experiments were performed as described [32 (link)]. Briefly, 129S6/SvEv WT donors were sacrificed and spleens were extracted. Spleens were cut into small pieces, dissociated, and cells were passed through 100 μm strainer. After red blood cell lysis (Pharm Lyse, BD) CD4⁺ cells were purified using negative selection kit and magnetic columns (Miltenyi Biotec). Cells were fluorescently labeled (anti-mouse CD4-PE and anti-mouse CD45-FITC, BD) and CD4⁺CD45^RBhigh population was sorted using BD FACSAria IIu at the University of Arizona Flow Cytometry Core Facility. Cells were counted and a suspension of 10⁶ cells/ml was prepared. A solution of 5x10⁵ cells in 500 μl PBS was intraperitoneally injected into Rag2^-/- (Rag/AT) and DKO (DKO/AT) mice. Control groups were injected with sterile PBS (Rag/PBS and DKO/PBS). Stool was collected on the day of injection and every two days afterwards. Body weight was recorded at the same time and monitored closely thereafter.

The density of second-generation BMSCs was adjusted to 2 × 10⁷ cells/mL. The control group received 50 µL of buffer, while the single-label group received 5 µL of hamster anti-CD29-AF647 (BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-CD90-PECyTM7 (BD Biosciences), mouse anti-CD106-PE (BD Biosciences), mouse anti-CD11b-V450 (BD Biosciences), or mouse anti-CD45-FITC (BD Biosciences) to each branch-flow sampling tube, after which 45 µL of buffer was added to each tube. The multicolor group received 5 µL of each antibody into a one-branch-flow sampling tube, and then, 25 µL of buffer was added. Next, 50 µL of cell suspension was added to each flow tube, the tubes were incubated at room temperature for 30 min and washed twice with staining buffer, and then, 500 µL of buffer was added to each tube for detection by flow cytometry (Beckman Coulter Life Sciences, Brea, CA, USA).