The aim of this procedure is to describe an experimental system to study mechanical transduction in fetal lung cells. This is accomplished by coding culture plates with extracellular matrix proteins. Then fetal mouse lung type two epithelial cells are isolated, followed by the isolation of fetal mouse fibroblasts.
The final step describes an in vitro system to provide mechanical stimulation to lung cells. Ultimately, results can be obtained that show an increase of type two cell differentiation through real-time PCR, western blot and fluorescence immunochemistry images. These method can help answer key questions in the mechano transduction field, such as fetal lung development and lung injury.
Demonstrating the procedure will be Julian gu, A technician from my laboratory While we're working in a laminar flow hood under sterile conditions makes 120 micrograms of laminin with 12 milliliters of cold sterile one XPBS per plate. Other extracellular matrix proteins may be used for coating. Consult the written protocol for details, then take a BioFlex untreated plate and add two milliliters of the laminate solution to each well to a final concentration of two micrograms per square centimeter.
Make sure the wells are completely covered by the solution. Fully cover the plate with plastic wrap and place on a flat surface in a four degree Celsius environment overnight to allow the laminate to absorb to the bottom of the wells. Coated plates can be stored under these conditions for at least a week while retaining good ECM function the next day.
Under sterile conditions, wash the wells three times with one XPBS. Then add one milliliter of 1%BSA in one XPBS to each. Well incubate at 37 degrees Celsius for one hour to block nonspecific binding sites on the membranes.
Then again, wash the wells three times with one XPBS. To remove unabsorbed proteins, add one milliliter of DMEM to each well of the plate. Then store the plate at 37 degrees Celsius until the fetal rodent lung type two epithelial cells are isolated and ready for plating.
The day before beginning the cell isolation procedure, assemble the screen cups with 130 and 15 micron nylon meshes and sterilize them by autoclaving. Also autoclave the same number of 150 milliliter glass peaks on the day of culture. Obtain fetal lungs from timed pregnant mouse on E 17 to 19 of gestation.
Transfer the tissue into a 50 milliliter centrifuge tube containing DMEA medium and place it on ice. Make fresh digestion buffer in a 50 milliliter centrifuge tube according to the recipe in the written protocol. And while working in a laminar flow hood filter, sterilize through a 0.2 micron syringe filter.
10 milliliters of this buffer is enough for the lung tissue obtained from approximately 20 mouse fetuses. Place the conical tube containing the digestion buffer in a water bath at 37 degrees Celsius. Asate the dium from the conical tube containing the fetal lung tissue and transfer the tissue into a sterile Petri dish using sterile scissors or a razor blade, mince the tissue into pieces less than one millimeter in size.
Then transfer the tissue into the conical tube containing the prewarm digestion buffer. Next, mechanically aid the digestion of the tissue by pipetting the fetal lung cell suspension up and down a hundred times each using pipettes of decreasing aperture size. After the digestion process is complete, centrifuge the homogenate at 1, 300 RPM for five minutes at room temperature.
Then carefully remove the supine by aspiration and resuspend the pellet containing the cells in 15 milliliters of DMEM plus 20%FBS. Next, retrieve the screen cups with 130 and 15 micron nylon meshes and place them on top of the sterile 150 milliliter beakers. Pipette the cell homogenate onto the 100 micron mesh.
Collect the filtrate and pipette this onto the 30 micron mesh. Wash the 30 micron mesh several times with fresh DMEM plus 10%FBS. Most of the type two cells clump together and do not pass through the mesh.
These washes remove non epithelial cells that may be attached to the clumps. Apply the filtrate from the 30 micron mesh to the 15 micron mesh screen cup. Again, wash several times as before.
This step recruits the type two cells that may have passed through the 30 micron mesh. Collect the clumped non-filtered cells from the 30 and 15 micron meshes for further type two cell enrichment. Retain the filtrate from the 15 micron mesh if fetal rodent lung fibroblasts are to be cultured.
Otherwise, discard this filtrate.Further. Purify the type two epithelial cell population by adding media and incubating the non-filtered cells from the 30 and 15 micron mesh cups in a 75 square centimeter tissue culture flask for 30 minutes after this incubation centrifuge the super name as before, de pellet the cells and then resuspend the pellet in two milliliters of serum free DMEM pro fetus pipette one milliliter of the cell suspension into each well of the laminate coated BioFlex six well plates at this point, the cells appear in clumps when observed under the microscope, incubate the plate in a tissue culture incubator set at 37 degrees Celsius and 5%carbon dioxide. A 24 hour incubation is optimal and a minimum of six hour incubation is required.
Before beginning mechanical strain experiments, take the filtrate from the 15 micron mesh and transfer to a 75 square centimeter tissue culture flask at 37 degrees Celsius for 30 to 60 minutes to allow fibroblast to adhere, aspirate, and discard the sate. Then replace the volume in the flask with serum free DMEM and incubate overnight. The next day, harvest the cells with 0.25%trypsin in 0.4 millimolar EDTA and plate them on fibronectin coated BioFlex plates incubate in a tissue culture incubator set at 37 degrees Celsius and 5%carbon dioxide, ideally for 24 hours before beginning mechanical strain experiments.
A minimum six hour incubation is required if a specific number of cells are required for the experiments. Type two cells are maintained in serum free DMEM 75 square centimeter flasks after isolation, and the next day cells are tryps inized counted, and then seated Monolayer should be no greater than 80%confluent before the initiation of the experiments. On the day of the mechanical stimulation experiment, the cell culture media is replaced with two milliliters of fresh serum free DMEM per well, and the plate is then mounted in a flex cell FX 5, 000 strain unit.
Next, apply EQU biaxial strain to the membranes. The regimen of strain varies depending on simulation of mechanical forces in vivo. As discussed in the written protocol, cells grown on non-ST stretched membranes.
Cultured in parallel are used as controls for the experiment. At the end of the experiment, monolayers can be processed to analyze changes in gene expression by real-time PCR or changes in protein abundance by western blot. In addition, monolayers can be fixed for immunochemistry experiments for this technique.
After fixation, astic membranes are cut out from the plates and mounted on glass slides using 10 to 20 microliters of water as a mounting agent before permeation and incubation with antibodies. The super datin from the experiment can also be used to investigate the presence of released substances such as growth factors or cytokines. This image shows parmal fixed E 18 fetal type two epithelial cells that were isolated as in this protocol and plated on BioFlex plates coded with laminin.
The cells were fixed and the photograph was taken. The day after the after non-ST stretch cells were plated. The purity of the cells was determined to be 90 plus or minus 5%by microscopic analysis of epithelial cell morphology and immunostaining for surfactant protein C.The following figures present data from fetal type two epithelial cells that were exposed to 5%cyclic strain at 40 cycles per minute for 16 hours.
This northern blot of surfactant protein C mRNA expression illustrates that strain induces type two cell differentiation using different ECM substrates. The plus minus signs represent exposure to strain or no exposure to strain respectively. The expression data are presented in the histogram as mean plus or minus SEM, the N equal three, and the asterisk indicates a P value of less than 0.05.
These fluorescence immunochemistry images show surfactant protein C protein levels in green. In fetal type two cells not exposed to mechanical strain on the left and exposed to mechanical strain on the right nuclei were counterstain with dapi, which appears blue. This histogram quantifies western blot results from three experiments showing that mechanical stretch increases the levels of surfactant protein C protein.
In this experiment, the N equal three and the asterisk indicates a P value of less than 0.05. After watching this video, you should have a good understanding of how to accelerate fetal lung cells and expose them to mechanical stretch.
