Anti cd44 fitc

Expression of cell surface molecules of canine AT-MSCs at passage 3 was evaluated by flow cytometry. Briefly, cells were harvested from cell flasks by enzymatic digestion with 0.25% trypsin-EDTA (Sigma-Aldrich). Next, cells were stained with conjugated antibodies (anti-CD45-Alexa 647, anti-CD19-FITC, anti-CD14-PE, anti-CD73-APC, anti-CD90-APC, anti-CD105-PE, and anti-CD44-FITC; all from eBioscience, San Diego, CA, USA) diluted in PBS containing 0.5% BSA. AT-MSCs were incubated for 20 min in the dark at 4°C. Following incubation, cells were washed with PBS and centrifuged at 300 ×g for 5 min. Cell pellets were resuspended with PBS for data acquisition using a BD FACSAria™ II flow cytometer. Data were analyzed using FlowJo software (FlowJo LCC, Ashland, OR, USA).

Cells exposed to indicated concentration of flubendazole for 72 hr, were trypsinized, washed twice with PBS by centrifugation (1,000 rpm, 5 min), and blocked (0.5% BSA/PBS) for 10 min. Collected cells were then incubated with anti-CD24 PE (eBioscience) and anti-CD44 FITC (eBioscience) or isotype controls antibodies according to manufacturer's instructions for 30 min at 4 °C protected from light. Then, cells were suspended in 1 ml PBS and subjected to flow cytometry analysis (Beckman). Side-scatter and forward-scatter profiles were used to eliminate cell doublets [51 (link)]. The statistical results of three independent experiments were presented.

Single-cell suspensions of spinal cord, spleen, and inguinal LNs were prepared as previously described [31 (link)]. The following antibodies were used: anti-CD4 PerCP-Cy5.5/APC/eFluor 450/PE-Cy7 (eBioscience, clone RM4-5), anti-CD45.1 FITC (eBioscience, clone A20), anti-CD45.2 PerCP-Cy5.5/APC (eBioscience, clone 104), and anti-CD44 FITC (eBioscience, clone IM7). For intracellular staining, cells were reactivated with culture media (negative control) or 5 μM MOG₃₅₋₅₅ peptide for 7 h with GolgiPlug (BD Biosciences) added for the final 4 h. The following intracellular antibodies were used in accordance with the manufacturer’s protocols: anti-IFNγ eFluor 450 (eBioscience, clone XMG1.2), anti-IL17A Alexa Fluor 647 (eBioscience, clone eBio17B7), anti-GM-CSF PE (BD Biosciences, clone MP1-22E9). A viability dye (Aqua, Life Technologies) was applied to exclude dead cells. Samples were acquired by using an LSRII flow cytometer (BD Biosciences) followed by data analysis using FlowJo version 9.x (Tree Star).

For the identification of GA-MSCs, flow cytometry and induced differentiation were performed as we described previously [14 (link)]. In brief, GA-MSCs were collected and stained with fluorochrome-conjugated antibodies, including anti-CD105-APC, anti-CD90-PerCP, anti-CD73-APC/Cy7, anti-CD44-FITC, anti-CD133-APC and anti-CD34-APC (all from eBioscience. USA) in the dark at 4 °C for 30 min. Then, the cells were centrifuged, resuspended and analyzed using a FACS flow cytometer (BD FACSCanto2, Biosciences). The data were collected and analyzed using FlowJo software (v10.6.2). GA-MSCs were differentiated into osteocytes, adipocytes, and chondrocytes by using ready-to-use differentiation media (all from Cyagen, China) following the manufacturer’s instructions. Adipogenic differentiation was evaluated by Oil Red O staining, osteogenic differentiation was evaluated by Alizarin Red staining, and chondrogenic differentiation was evaluated by Alcian Blue staining. The stain results were observed with an inverted phase contrast microscope (Olympus IX73), and photographs were taken with a digital camera using Image-Pro Plus 6.0 software.

To analyze cell surface proteins levels, 5 × 10⁵ lymphocytes were labeled with the following mAntibodies: anti-B220 FITC, anti-CD4 PE, anti-IFNγ PE (BD Pharmingen™), anti-CD122 PE, anti-CD127 PE, anti-CD25 APC, anti-CD25.AF488, anti-CD3 APC, anti-CD44 FITC, anti-CD62L PE, anti-CD62L PErCP.Cy5.5, anti-CD8 PerCP.Cy5.5, anti-Granzyme B FITC, anti-IgG2a K FITC, anti-IgG2a K PE, anti-Klrg1 FITC (all eBioscience™). For intracellular staining, 1 × 10⁶ cells/ml were stimulated in vitro for 6 h with 10 nM of Phorbol 12-myristate 13-acetate (PMA) plus 1 μM of ionomycin (both from Calbiochem^®). Brefeldin A (1:1000; BD Pharmingen™) was added to the culture for the last 2 h. Cells were harvested and stained with anti-CD44 APC and anti-CD62L PErCP.Cy5.5 antibodies. Then, the cells were fixed, permeabilized, and stained with anti-Granzyme B FITC, anti-IFN-γ PE or anti-IL-2 PE antibodies and analyzed by flow cytometry on a FACSCalibur. For the memory subtype analysis, day 10 memory cells generated in vitro were sorted using a MoFlo Cell Sorter (Beckman Coulter, Inc) based on the CD62L level. All the flow cytometry data were analyzed using FlowJo^® software.

Six-month-old/22-month-old rats were sacrificed by euthanized by exsanguination under 2% isoflurane anaesthesia. Under sterile conditions, 1 mL of bone marrow was extracted from the femur and tibia using a syringe prefilled with 2 mL of heparin for anticoagulation. Bone marrow mononuclear cells were separated by gradient density centrifugation and cultured in osteogenic differentiation medium of Wistar rat BMSCs (Cyagen Biosciences, RAWMX-90021, Santa Clara, CA) supplemented with 20% foetal bovine serum (HyClone, Logan, UT), 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in a 5% CO₂ atmosphere (Thermo, Waltham, MA). Forty-eight hours later, nonadherent cells were removed, and the adherent cells were cultured and expanded in another flask. After 2–3 weeks of continuous culture, homogenous rBMSCs from young and old animals were obtained and identified by immunofluorescence.
The identification of rBMSCs was performed by immunofluorescence using a fluorescently labelled antibody. rBMSCs were fixed in 4% (w/v) paraformaldehyde in PBS for 20 min and then incubated with anti-CD29-FITC (eBioscience, #11-0291-82, Shanghai, China), anti-CD44-FITC (eBioscience, #MA5-17522, Shanghai, China) and anti-CD45-FITC (eBioscience, #11-0461-82, Shanghai, China). Visual analysis was performed with an Olympus fluorescence microscope (Olympus, CX71, Tokyo, Japan) at ×400 magnification.