The overall goal of the following experiment is to determine the number of neutrophils that have crossed an epithelial cell monolayer in response to infection. This is achieved by collagen coding transwells before carefully cultivating epithelial cell monolayers using the inverted transwell filter maneuver. As a second step bacterial pathogen is cultured in order to infect the apical surface of the epithelial monolayer grown on the transwell filters, which causes the epithelial cells to produce endogenous neutrophil chemo attractants.
Next neutrophils isolated from whole blood are added to the basolateral side of infected epithelial monolayers grown on transwell filters for assaying migration of neutrophils from the basolateral to the apical side. Results are obtained that show a significant increase in the number of trans epithelial migrating neutrophils observed in response to infected epithelial monolayers based on quantification of myelo peroxidase from migrated neutrophils. Visual demonstration of this method is critical as the inverted transwell filter maneuver to grow and infect epithelial barriers is unconventional and therefore can be less approachable for unfamiliar research groups to establish in their laboratory.
The implications of this technique extend toward therapy of airway inflammatory diseases involving infection such as pneumonia or cystic fibrosis, because novel treatments can be developed and tested to decrease the amount of neutrophil trans epithelial migration, which may mitigate the deleterious effects of overzealous neutrophilic, breach of mucosal barriers. Along with Mark, who is a clinical fellow in my lab, demonstrating the procedure will be senior technician Wahi Preza and postdoctoral fellow Michael Pesos. To begin remove 0.33 square centimeter growth area, Transwells with a pore size of three micrometers from the packaging and place the 24 well plate containing 12 transwells upside down inside the hood.
Lift off the bottom plate and place aside. Transwells will now be upside down. Resting atop the lid of the 24 well plate flame, a hemostat for sterility and let cool.
Using the sterile hemostat transfer transwells to a 150 by 25 millimeter Petri dish, keeping them in the inverted position, pipette 70 microliters of a prepared 30 microgram per milliliter collagen solution in ethanol onto the filter membrane of each inverted transwell surface Tension should hold this volume of solution in place as there are no walls around the filter membrane when accessing the surface of the underside. Be careful that the collagen solution does not drip down the side of the transwell and avoid moving transwells during the coating procedure. Leave the lid of the Petri dish off for a minimum of four hours to allow the ethanol to evaporate To maintain sterility during this process.
Keep the hood fan and the ultraviolet light on after packaging epithelial cells as described in the text protocol. Add 70 microliters of the Resus suspended cell solution from a 15 milliliter tube to each inverted collagen coated transwell. Mix the cell suspension by gently inverting the tube periodically to prevent cells from settling to the bottom of the 15 milliliter tube while seeding the transwells.
Once all inverted transwells have been seeded with epithelial cells, replace the Petri dish cover and carefully place the cells in the tissue culture incubator the following day. Add one milliliter of media into each well of the original sterile 24 Well plate and use a sterilized hemostat to flip each inverted trans well into each well containing one milliliter of media. Add 0.2 milliliters of media into the top chamber of each trans well and return to the tissue culture incubator.
Remove the 24 well plate supporting epithelial layers that have been cultured on the underside of Transwell filter membranes for at least eight days from the incubator. Grasp the edge of each trans well with a hemostat and lift it out of the 24 well plate. Invert the transwell over a waste bucket to discard culture media from the inner chamber of the Transwell.
Dip each transwell into a wash beaker containing Hank's balanced salt solution with calcium and magnesium or Hank's Plus to fill the inner chamber of the Transwell. Then discard the fluid from the inner chamber into a waste bucket. Repeat this step for a second.
Wash in Hank's plus after two washes. Place the Transwells into a new 24 well plate containing one milliliter of Hank's Plus. In each well observe the top chamber of each transwell to assess epithelial cell layer integrity.
Hank's Plus should not leak into and fill the top chamber of the Transwell when placed in the well of a 24 well plate containing one milliliter of Hank's Plus. After carefully observing each transwell, add 0.2 milliliters of Hanks Plus into the top chamber. Place the plate in the incubator for 30 to 60 minutes to equilibrate epithelial cell layers to perform the neutrophil trans epithelial migration assay.
Grasp each transwell with a hemostat from the 24 well plate of Wash Transwells equilibrated in Hank's Plus and discard the fluid in the top well into a waste bucket. Place each transwell upside down in a 150 by 25 millimeter Petri dish to six of the inverted transwells. Add 25 microliters of Hanks plus to three of the inverted transwells.
Infect the cells with 25 microliters Pseudomonas, arosa or Pao one diluted in Hanks plus prepared as described in the text protocol to the final three Inverted transwells infect the cells with 25 microliters of e Coli, K 12, or mc 1000 diluted in Hanks Plus also prepared as described in the text protocol after incubating at 37 degrees Celsius and 5%carbon dioxide in a humidified chamber for 60 minutes. Wash the transwells by grasping with a hemostat and flipping them back over into a 24 well wash plate that contains one milliliter of Hank's plus. In the bottom chambers.
Add 0.2 milliliters of Hank's plus to the top chambers. Grasp the Transwell with a hemostat and remove it from the first wash plate. Discard the fluid from the top chamber into a waste bucket.
Prior to placing the Transwells into a second 24 well wash plate containing one milliliter of Hanks plus add 0.2 milliliters of Hanks plus to the top chamber. Repeat this step one more time for a total of three washes. After the third wash, discard the fluid from the top chambers of the trans wells into a waste bucket using a hemostat place three of the uninfected trans wells into three wells of the 24 Well migration plate containing one milliliter of Hanks Plus, which represents the negative control pipette 10 microliters of a 10 micromolar solution of FMLP into each of three wells of the 24 Well migration plate containing one milliliter of Hanks plus place three of the uninfected trans wells into three wells of the 24 Well migration plate containing 100 Nanomolar FMLP, which represents a positive control for the ability of isolated neutrophils to migrate.
Then place the three trans wells infected with PAO one and the three trans wells infected with mc 1000 into three corresponding wells of the 24 Well migration plate containing one milliliter of Hanks plus add 0.1 milliliter of Hanks plus to the top chamber of every trans well on top of the 0.1 milliliter Hanks plus in each trans. Well carefully add 20 microliters of the neutrophil suspension prepared as described in the text protocol. Incubate for two hours to allow trans epithelial migration of the neutrophils.
After two hours of migration, lift the transwells by grasping with a hemostat and tap gently against the inside walls of the 24 well migration plate. Before discarding the trans wells, assess the migration of neutrophils grossly in the wells by viewing wells of the 24 well migration plate under an inverted microscope using the 10 x objective. Then add 50 microliters of 10%Triton X 100 to each well used in the 24 well migration plate, each standard and the blank.
Rotate the 24 well migration plate standards and blank at low speed for 20 minutes. At four degrees Celsius. Add 50 microliters of citrate buffer.
Two each well used in the 24 well migration plate to each standard and to the blank thoroughly. Mix the contents in each sample well by pipetting the solution up and down at least five times. Then transfer 100 microliters of each sample well in duplicate to a 96 well plate transfer, 100 microliters of each standard and the blank to the 96 well plate.
After mixing, add 50 microliters of 30%hydrogen peroxide to the A BTS solution and vortex. Then add 100 microliters of A BTS substrate solution to each sample. On the 96 well plate, allow the plate to develop for five to 10 minutes in the dark.
Observe the development of green color in the wells of the plate containing lyse neutrophils before reading at a wavelength of 405 nanometers. Using a microplate reader to assess neutrophil trans epithelial migration neutrophils that have migrated fully across the epithelial layer to the apical chamber can be viewed in the bottom well of the 24 well migration plate as visualized here using an inverted light microscope. Very few neutrophils were observed to migrate across an uninfected epithelial layer without imposed chemotactic gradient and represent background levels in the assay.
In contrast, an abundance of transmi neutrophils were apparent when an FMLP gradient was provided. Infection of the epithelium with non-pathogenic e coli mc 1000 resulted in few visible trans migrated neutrophils, whereas many trans migrated neutrophils were observable when epithelial layers were infected with the lung pathogenic pseudomonas aerogen. The neutrophils that have migrated in the experiment are quantified by measuring their myelo peroxidase activity.
The number of neutrophils positively correlates with the amount of peroxidase activity measured following lysis of neutrophils with values exhibiting a linear relationship in the range of neutrophil numbers selected for the standard curve. A significant number of neutrophils migrate across epithelial layers in response to A provided FMLP gradient, or in response to an epithelial layer infected with PAO one. The number of neutrophils that migrate in the absence of stimuli or following apical epithelial infection with non-pathogenic e coli mc 1000 is below the limit of detection for the assay.
Once mastered, this technique can be done in five hours for collagen coating and seeding the epithelial cells on the transwell filters, followed by at least one week to allow the epithelial cells to grow. Once epithelial monolayers are ready. The migration assay can be accomplished in five hours, including the isolation of neutrophils and the preparation of bacteria if it is performed properly.
While attempting this procedure, it's important to remember to prepare the transwell filters under sterile conditions to prevent contamination. This procedure can be adapted in order to answer additional questions related to this inflammatory process. Analysis of the migrated cells, the epithelium, the supine, or even the pathogen can be performed.
