Half of the MNCs were resuspended in the Endocult medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco/Invitrogen, Carlsbad, CA, USA). The suspension was seeded into one well of 6-well fibronectin-coated plates (BD Biosciences) and incubated for two days at 37℃, 5% CO2, and 95% humidity. Then, the non-adherent cells were collected and replated onto 24-well fibronectin-coated plates (BD Biosciences). After 5 days, the culture medium was removed and the plates were washed with phosphate buffered saline. To increase the accuracy, the tests were performed in duplicate and the colonies were counted by two independent expert technicians.
24 well fibronectin coated plates
Manufactured by BD
Sourced in United States
The 24-well fibronectin-coated plates are a type of cell culture equipment designed for in vitro cell-based experiments. The plates feature a 24-well format and have a surface that is pre-coated with the extracellular matrix protein fibronectin. This coating promotes cell attachment and growth, making the plates suitable for a variety of cell culture applications.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fibronectin coated plate, by BD (1 mentions)
Endocult medium, by STEMCELL (1 mentions)
Uea 1, by Merck Group (1 mentions)
Dildl, by Thermo Fisher Scientific (1 mentions)
Total rna extraction kit, by Norgen Biotek (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Proline, by Merck Group (1 mentions)
Tgf β3, by R&D Systems (1 mentions)
Formalin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Na pyruvate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (1 mentions)
Its1 mix, by BD (1 mentions)
4 protocols using 24 well fibronectin coated plates
1
Isolation and culture of MNCs
2
Isolation and Characterization of Circulating Angiogenic Cells
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CACs were isolated from approximately 14 ml heparinized peripheral blood. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation with Ficoll (Ficoll-Paque™ PLUS, GE Healthcare Bio-Sciences Uppsala, Sweden) within 2 h of collection. Then 5×106 PBMCs were plated on 24-well fibronectin-coated plates (BD Biosciences, Mountain View, CA, USA) and maintained in endothelial basal medium (EBM; Clonetics-Lonza, Walkersville, MD USA) supplemented with EGM SingleQuots and 20 % fetal calf serum for 4 days. After 4 days in culture, non-adherent cells were removed by washing in PBS, whereas adherent cells were lysed directly in the culture wells.
The CAC phenotype was confirmed by cellular uptake of acetylated LDL (DiI-acLDL) and binding of FITC-conjugated lectin from Ulex europaeus (UEA-1) by fluorescence microscopy. Briefly, to detect DiLDL uptake, cells were incubated with DiLDL (2.4 μg/ml) (Molecular Probes, Eugene, OR, USA) at 37°C for 2 h. To detect UEA-1 binding, cells were fixed with 2 % paraformaldehyde for 15 min and incubated with FITC-labeled UEA-1 (10 μg/ml) (UEA-1, Sigma, St. Louis, MO, USA) for 1 h. Double-stained cells positive for both UEA-1 and DiLDL were regarded as CACs. RNA was purified according to the instruction manual of the Total RNA Extraction kit (Norgen Biotek).
The CAC phenotype was confirmed by cellular uptake of acetylated LDL (DiI-acLDL) and binding of FITC-conjugated lectin from Ulex europaeus (UEA-1) by fluorescence microscopy. Briefly, to detect DiLDL uptake, cells were incubated with DiLDL (2.4 μg/ml) (Molecular Probes, Eugene, OR, USA) at 37°C for 2 h. To detect UEA-1 binding, cells were fixed with 2 % paraformaldehyde for 15 min and incubated with FITC-labeled UEA-1 (10 μg/ml) (UEA-1, Sigma, St. Louis, MO, USA) for 1 h. Double-stained cells positive for both UEA-1 and DiLDL were regarded as CACs. RNA was purified according to the instruction manual of the Total RNA Extraction kit (Norgen Biotek).
3
Isolation and Culture of Rat EPCs
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EPC culture and characterization was performed as previously described (16 (link),17 (link)). In brief, mononuclear cells were isolated from the spleens of Sprague Dawley (SD) rats (n=6; male; age, 12 weeks; weight, 320–380 g; Third Military Medical University, Chongqing, China). The cells were counted and seeded on fibronectin-coated 24-well plates (BD Biosciences) at 1×106 cells/well and then grown in EC basal medium 2 (EBM-2; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 5% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) containing an EPC growth cytokine cocktail (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO2 incubator at 37°C. After 3 days, any non-adherent cells were removed by rinsing three times with PBS. Thereafter, the culture medium was changed every two days.
4
Two-Dimensional Chondrogenic Induction
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Two-dimensional chondrogenic induction was performed as previously described [30] (link). Briefly, cells (1.5×105) that induced hMSCs were suspended in 5 µl of chondrogenic medium (DMEM: F12 (Invitrogen), 1% (v/v) ITS1 mix (BD), 0.17 mM AA2P, 0.35 mM Proline (Sigma), 0.1 mM dexamethasone (Sigma), 0.15% (v/v) glucose (Sigma), 1 mM Na-pyruvate (Invitrogen), 2 mM GlutaMax, and 0.05 mM MTG supplemented with 40 ng/ml PDGF-BB and 1% (v/v) FBS (Nichirei)), and were subsequently transferred to fibronectin-coated 24-well plates (BD). A total of 1 ml of the chondrogenic medium was added after 1 hour. TGFβ3 (R&D) was subsequently added at 10 ng/ml on days 3 to 6, and BMP4 was added to a concentration of 50 ng/ml on day 10. Micromass cultures were maintained at 37°C under 5% CO2 and 5% O2 for 16 days. Differentiation properties were confirmed by Alcian Blue staining. Briefly, induced cells were fixed for 30 minutes with 10% formalin (Sigma) and rinsed with PBS. These cells were then stained overnight with Alcian Blue solution (1% Alcian Blue (MUTO PURE CHEMICAL CO., LTD, Tokyo, Japan) in 3% glacial acetic and 1% HCl, pH 1) and destained with the acetic acid solution.
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