At P2, a sub-culture of cells from each cord was characterized through cell surface marker identification via flow cytometry on a MoFlo XDF fluorescent activated cell sorter (FACS) (Beckman Coulter, Brea, CA). hWJCs were characterized using the following antibodies and secondary antibodies: STRO-1 Mouse IgM (2.5:200) (1 mg per mL; R&D Systems, Minneapolis, MN); Alexa Fluor 568® Rabbit Anti-Mouse IgG (2:200) (2 mg per mL; Life Technologies); CD105 Mouse IgG (2.5:200) (1 mg per mL; R&D Systems); Qdot® 525 donkey anti-mouse IgG (2:200) (1 μM; Life Technologies); Human CD45 pre-conjugated to Qdot® 800 (2:200) (Life Technologies); Human CD73 pre-conjugated to FITC (5:200) (BD Biosciences, San Jose, CA); Human CD34 pre-conjugated to Brilliant Violet (5:200) (BD Biosciences); Human CD90 pre-conjugated to APC (5:200) (BD Biosciences). 20,000 events were recorded for each sample. Positive identification of cell markers was defined as fluorescent emission that exceeded the fluorescent threshold of cells stained with corresponding isotype (negative) controls. The isotype controls used in these studies were Rabbit IgG Alexa Fluor 568, Donkey IgG Qdot 525, IgG2 Qdot 800 (all from Life Technologies), and IgG1 FITC, IgG1 Brilliant Violet, and IgG1 APC (all from BD Biosciences). The cell characterization experiments were repeated three times for each cord.
Moflo xdf
Manufactured by Beckman Coulter
Sourced in United States, China
The MoFlo XDF is a high-performance cell sorter manufactured by Beckman Coulter. It is designed for advanced flow cytometry applications. The MoFlo XDF offers precise sorting capabilities and high-speed performance.
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Lab products found in correlation
3 protocols using moflo xdf
1
Characterization of Wharton's Jelly Cells
2
Isolation and Sorting of RANK+ Mouse MSPCs
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For flow cytometric analysis and sorting of mouse RANK+ MSPCs from femoral metaphysis, we dissected the femora free of soft tissues from mice offspring at 12 weeks old. The epiphysis was removed, and the bones were cut into small pieces and digested in the protease solution for 20 min. Cells within the supernatant were collected for flow cytometry. Cell numbers were determined after removal of red blood cells with ACK Lysis Buffer (CS0001, Leagene, China). Cells were then sorted according to side scatter and RANK-PE fluorescence at >103 log Fl-1 (RANK-PE) fluorescence. FACS was performed using a 5-laser Beckman FACS system (MoFlo XDF, Beckman, United States). Flow cytometric analyses were performed using FlowJo software (BD Life Sciences, San Jose, CA, United States). The primary antibody used was RANK-PE (12-6612-82, Thermo Fisher Scientific, 5 μL/106 cells).
3
Flow Cytometry Protocol for S. meliloti Cell Analysis
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De Nisco’s flow cytometry protocol was used [33 (link)]. The cells from 4 mL of S. meliloti cultures were collected by centrifugation (6000 rpm, 5 min, 4 °C), and washed twice with a 0.85% NaCl solution (stored at 4 °C). Then, 250 μL of cell suspension was mixed with 1 mL of 100% ethanol to fixation. The fixed cells were collected by centrifugation (6000 rpm, 3 min), and incubated in 1 mL of 50 mM sodium citrate buffer containing 4 μg/mL RNase A at 50 °C for 1.5 h. Then, 1 μL of 10 μM SYTOX Green dye (Sigma, Shanghai, China) was added to each sample. Each sample was assessed using a MoFlo XDF (Beckman Coulter, Suzhou, China) flow cytometer, and the results were analyzed by Summit 5.1 software (Beckman Coulter, Suzhou, China).
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