Pe conjugated anti α sma

Single cells were isolated from both normal and diseased kidneys using enzyme-digestion and analyzed by flow cytometry as previously described.⁴⁷ After permeabilization, cells were incubated with FITC-conjugated rat anti-mouse F4/80 (eBioscience) or rat anti-mouse CD11b (Serotec) followed by Cy5-conjugated goat anti-rat IgG (Millpore), PE-conjugated anti-α-SMA (R & D, Minneapolis, MN, USA), FITC-labeled rat anti-mouse CD206 (Serotec) or rabbit anti-CX3CR1(Prosci Inc). Collagen I was stained with rabbit anti-mouse collagen I (Millpore) followed by the Cy5 labeled goat anti-rabbit Ig (Invitrogen). Cells incubated with isotype-matched irrelevant control antibodies and unstained cells were used as negative controls. Cells were detected by a FACS Calibur flow cytometer (BD Biosciences) and analyzed using Cellquest software.

Single cells were isolated from both normal and fibrotic kidneys using enzyme-digestion and analyzed by flow cytometry as previously described.^{17 (link)} After permeabilization with Fixation/Permeabilization buffer kit (eBioscience, San Diego, CA, USA), cells were incubated with FITC-conjugated rat anti-mouse F4/80 (eBioscience) followed by Cy5-conjugated goat anti-rat IgG (Millpore), PE-conjugated anti-α-SMA (R&D, Minneapolis, MN, USA), FITC-labeled rat anti-mouse CD206 (Serotec). Collagen I was stained with rabbit anti-mouse collagen I (Millpore) followed by the Cy5-labeled goat anti-rabbit IgG (Invitrogen). Cells stained with isotype-matched irrelevant control antibodies and unstained cells were used as negative controls. Cells were detected by a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed by Cellquest software (BD Biosciences).