Pe conjugated antibodies against cd44

The hAFSCs were obtained from freshly collected amniotic fluid by routine amniocentesis from second-trimester healthy pregnant donors. Cells were cultured in StemPro® MSC SFM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C with 5% CO₂. The specific surface antigens of hAFSCs were characterized using flow cytometry according to our previous work^{13 (link)–15 (link)}. The cells in culture were trypsinized and stained with phycoerythrin (PE)-conjugated antibodies against CD44, CD45, CD73, CD90, CD105 and CD117 (BD PharMingen, San Diego, CA, USA). Thereafter, the cells were analyzed using Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Passage 6–8 hAFSCs were collected and prepared to a final concentration of 1 × 10⁶ cells/0.3 mL in PBS. In the hAFSCs-treated groups, 1 × 10⁶ collected hAFSCs were injected into 5 sites of bladder in each rat (anterior, posterior, bilateral, and dome) under inhalation anesthesia according to our previous method^{14 (link),15 (link)}. The treatment dose of hAFSCs was determined according to our previous study that used hAFSCs to treat bladder dysfunction after focal cerebral ischemia in rats^{15 (link)}.

hAFSCs were obtained using freshly collected amniotic fluid by routine amniocentesis from healthy pregnant donors in 15–20 gestational weeks. Institutional review board of our hospital approved this study (No. 201800452A3). Cells were cultured in StemPro® MSC serum‐free medium supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA, USA) and incubated at 37°C with 5% carbon dioxide. Culture medium was changed every 3–4 days. Specific surface antigens of hAFSCs were characterized by flow cytometry analyses as shown in our previous work.¹⁴ Cultured cells were trypsinized and stained with phycoerythrin (PE)‐conjugated antibodies against CD44, CD73, CD90, CD105, CD117 and CD45 (BD PharMingen, CA, USA). Thereafter, cells were analysed using Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Passage 4–6 hAFSCs were collected and prepared to a final concentration of 5 × 10⁶ cells/0.3 ml in phosphate buffer solution (PBS). In hAFSCs‐treated groups, 5 × 10⁶ hAFSCs were administrated via tail vein at 3 h after MCAO. In DM + MCAO groups, 0.3 ml PBS were injected into tail vein at 3 h after MCAO. The timing of hAFSCs injection was based on our previous study.¹⁴ The treatment dose of hAFSCs was determined according to our previous studies that used stem cells to treat stroke in T1DM rats.¹³, ¹⁴