This is approximately the strength of a refrigerator magnet, and there was no difference between MNP-loaded and untreated HCECs (p=0.134; Fig. Transplant and Sulfo-NHS-LC-Biotin Research (Tampa, FL), the Florida Lions Vision Lender (Miami, FL) and the National Disease Research Interchange (NDRI, Philadelphia, PA). Main cultures of HCECs were purified and expanded following the method explained by Zhu and Joyce (2004) with some modifications. In brief, corneas were rinsed 3 times in M199 with gentamicin 50 g/l (Gibco-Invitrogen, Carlsbad, CA). Endothelium pieces attached to Descemets membrane were cautiously stripped off with forceps under a dissection microscope and incubated in growth medium made up of OptiMEM-I (Gibco BRL-Life Technologies, Rockville, MD), 8% FBS (Thermoscientific-Hyclone, Logan, UT), 5 ng/mL EGF, 20 ng/mL NGF, 100 g/mL bovine pituitary extract (Biomedical Technologies, Stoughton, MA), 20 g/mL ascorbic acid (Sigma, St. Louis, MI), 200 mg/L calcium chloride (Invitrogen-Gibco, Carlsbad, CA), 0.08% chondroitin sulfate (Sigma, St. Louis, MI), 50 g/mL gentamicin, and antibiotic/antimycotic answer diluted 1:100 (Invitrogen-Gibco, Carlsbad, CA) overnight at 37C and 5% CO2 for stabilization. The next day, the tissue was centrifuged at 931 RCF for 6 moments, washed in HBSS (Gibco BRL-Life Technologies, Rockville, MD) and incubated in 0.02% EDTA (Sigma, St. Louis, MI) for 1 hour at 37C. Cells were released by mechanical disruption by passing the tissue 15C20 occasions through a glass pipette. Cells were pelleted and resuspended in growth medium. Isolated cells and pieces of Descemets membrane from a single cornea were FGF-18 plated in one well of 6-well or 12-well tissue culture plates pre-coated with FNC Covering Mix (US Biological, Salem, MA) for 15 minutes at room heat. All cultures were incubated at 37C in a 5% CO2, humidified atmosphere. Media was changed every other day. Cell passaging was performed after cultures reached confluency, using trypsin to split the culture in a 1:2 to 1 1:3 ratio. Addition of MNPs to HCECs in Culture Rat anti-mouse IgG1 superparamagnetic MACS MicroBeads (150 L, 50 nm diameter; Miltenyi Biotec) were centrifuged at 6010 RCF for 10 minutes at 4C, the supernatant was removed, and MNPs were washed with 500 L of 0.02% sterile filtered BSA in D-PBS. This was centrifuged again at 6010 RCF for 10 minutes at 4C, the supernatant removed, and the nanoparticles resuspended in 150 L of 0.02% BSA in D-PBS. This was placed in a bath sonicator (Fisher Scientific FS 15, Pittsburgh, PA) for 4 moments at room temperature. The desired volume of MNPs (e.g. 1, 3, 10, 100, or 1000 L) was then softly delivered, in a spiral motion, to a single well of HCECs that experienced reached confluence in a 6-well plate (700,000 to 1 1,200,000 HCECs); the dish was then lightly shaken. HCECs were incubated with the MNPs for 24 hours at 37C in adherent culture, except for time-dependence experiments where they were incubated for varying time intervals, as Sulfo-NHS-LC-Biotin noted. Magnetic-HCECs were harvested with 0.05% trypsin (Invitrogen-Gibco, Carlsbad, CA) incubated for 5 minutes at 37C. Immunostaining For immunostaining of the tight junctions of MNP-loaded HCECs, 50,000 cells on passage 3 were seeded on FNC-coated Sulfo-NHS-LC-Biotin glass coverslips (Carolina Biological Supply Co., Burlington, NC). MNPs were added as explained above either at the time of plating or 4 days later, and all cells were harvested after 5DIV. Thus, HCECs were fixed after an overnight or 5-day incubation with MNPs, in 3% paraformaldehyde in PBS for 20 moments at room temperature, rinsed three times with PBS and permeabiliized with 0.05% Triton X-100 in PBS for 3 minutes. After washing twice with PBS and next with 5% non-fat dry milk in PBS, main antibody (rabbit anti-ZO-1; Invitrogen, Carlsbad, CA 10 g/ml) was diluted in 5% milk buffer and incubated for one hour at room heat. One coverslip was incubated with 5% non-fat dry milk in PBS only, as a negative control. Next, after three washes in 5% milk in PBS, coverslips were incubated in the dark with goat anti-rabbit-Alexa 546-conjugated secondary antibody (1:1000; Life Technologies, Grand Island, NY) in 5% milk in PBS for 30 minutes at room heat. After two washes in 5% milk in PBS and three washes in PBS, coverslips were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA) and imaged with an upright fluorescent microscope Observer Z1 (Carl Zeiss Microscopy, GmbH, Germany). Four 40 images per coverslip were stitched together using Photoshop (Adobe Systems Inc, San Jose, CA) to offer a far.
- For three of the four experiments included in Fig
- Comparative gene expression levels were analyzed using the two 2?Cq technique (27)
- Finally, MSCs in ePL or FBS, following 28 days of chondrogenic media exposure, showed increased proteoglycans simply by Alcian Blue staining weighed against the undifferentiated group (Fig
- Phenotypic identity of MSC was verified using flow cytometry for the current presence of CD44, CD117 and CD90, as described by all of us previously
- Data Availability StatementThe datasets used and/or analyzed during the current study are available from your corresponding author on reasonable request