This protocol uses three approaches to determine if cigarette smoke affects pseudomonas bacterial load in lung epithelial cells. This method can be expanded to endothelial cells or other cell taps. Preparation of the cigarette smoke extraction, bacterial infection and the determination with a bacterial load are well described in detail in this video.
This procedure is very easy to follow for the new users. Draw 10 milliliters of serum-free cell culture medium into a 60 milliliter syringe. Attach the narrow end of a trim to one milliliter pipette tip to the nozzle of the syringe, as an adapter to hold the cigarette.
Remove the filter from the cigarette and attach the cigarette to the adapter. No more than 30 minutes before performing the assay combust the cigarette and draw 40 milliliters of smoke containing air into the syringe. Mix the smoke with the medium by shaking the syringe vigorously.
Repeat the drawing process until the cigarette is completely burned out, which will require about 11 draws in approximately seven minutes. To remove micro-organisms and insoluble particles from the medium, filter it through a 0.22 micron filter. Then, transfer the medium to a closed sterile tube.
Inoculate a tryptic soy broth or TSB auger plate with a selected pseudomonas strain. Incubate the plate overnight at 37 degrees Celsius. Prepare a tube containing 20 milliliters of TSB with 5%glycerol as the carbon source.
Collect a smear from the auger plate and inoculate the TSB. Incubate the pseudomonas suspension in a 37 degrees Celsius shaker at 200 RPM, for approximately one hour, until the optical density at 600 nanometers is 0.6. After culturing lung epithelial cells as described in the manuscript, add one milliliter of 0.25%trypsin to the cells.
After approximately five minutes, when the cells have completely detached from the bottom of the plate, add 10 milliliters of complete Heights medium to neutralize the trypsin. Transfer the cells to a 15 milliliter tube. Then, centrifuge the cells for five minutes at four degrees Celsius and 300 times G.Remove and discard the supernatant.
Then, re-suspend the cells into milliliters of Heights medium with 10%FBS. Pipette 10 microliters of the epithelial cell suspension onto a new plate. Use an automated cell counter to obtain the cell concentration.
Plate the lung epithelial cells at a concentration of three times 10 to the fifth cells per milliliter, in a total volume of two milliliters of medium per well. Incubate the plates overnight. When the cells reach approximately 80%confluency, replace the medium with Heights medium, plus 1%FBS.
Then, add 4%CSE to the wells and incubate the plates for three hours. Add a pseudomonas to each well of CSE-treated lung epithelial cells. Then, incubate the plates for one hour at 37 degrees Celsius with 5%carbon dioxide.
Next, replace the medium in the six well plates with two milliliters of fresh Heights medium, containing 4%CSE and 100 micrograms per milliliter of gentamycin. After incubating the plates for one hour at 37 degrees Celsius with 5%carbon dioxide, aspirate the medium from the wells. Wash the gentamycin-treated cells, twice, with two milliliters of cold PBS.
Bacteria load in the lung epithelial cells can then be detected by the drop plate method, qRT-PCR, or flow cytometry. Add one milliliter of cell lysis buffer to each well of lung epithelial cells. Repair serial dilutions of the cell lysate, ranging from one to 10 to one to 10, 000.
Then inoculate the TSB auger plate. After 16 hours of incubation, determine how many colony forming units were present in the lung epithelial cells, by counting the number of bacterial colonies. Add 0.35 milliliters of the guanidinium thiocyanate lysis buffer to each well of lung epithelial cells.
Collect the cells with a cell scraper. Pipette the lysate into a micro-centrifuge tube and mix gently with the pipette. Add 0.35 milliliters of freshly prepared to 70%ethanol to the lysate and mix well.
Transfer all samples to a spin column placed in a two milliliter collection tube. Centrifuge the collection tube for 30 seconds at 10, 000 times G and 20 to 25 degrees Celsius. Discard the buffer in the collection tube and wash the column with 0.7 milliliters of wash buffer.
Centrifuge the column at 10, 000 times G for 30 seconds. Wash the column twice with 0.5 milliliters of buffer. Repeat the centrifugation at 10, 000 times G for two minutes.
Place the column into a new 1.5 milliliter collection tube and add 30 to 50 microliters of RNase-free water. After centrifuging the tube at 10, 000 times G for one minute, collect the flow through and measure the RNA concentration. To prepare for a 20 microliter reverse transcription reaction makes one microgram of total RNA with 10 microliters of reaction buffer, one microliter of reverse transcriptase and RNase-free water.
Conduct the reverse transcription reaction at 37 degrees Celsius for one hour and then at 95 degrees Celsius for five minutes, according to the manufacturer's protocol. Then mix one microliter of each cDNA sample with five microliters of the master mix containing cyber dye and one microliter of each 200 nanomolar specific primer. Add water for a total volume of 20 microliters.
Perform PCR analysis, according to the manufacturer's recommendations. And then use the comparative CT method to determine the expression. In BEAS-2B cells, viability did not change considerably when cells were treated with 4%CSE for three hours.
One hour as pseudomonas infection also did not substantially affect viability. Finally, they use of gentamycin to kill pseudomonas in the culture medium didn't have a statistically significant effect on cell viability. As measured by the drop plate method, bacterial infection in BEAS-2B cells was substantially increased by treatment with CSE.
The same was true for CSE treatment of HSAEC. Bacterial infection of CSE-treated BEAS-2B cells was also assessed by using RT-PCR to quantify the amount of 16S rRNA present, which indicated an increase in Pseudomonas aeruginosa. The BEAS-2B cells treated with CSE and infected with Pseudomonas fluorescens Migula were analyzed by flow cytometry at 509 nanometers.
The result also indicated that CSE treatment increases bacterial infection. Results from fluorescent microscopy showed that GFP-labeled bacteria co-localized with BEAS-2B cells in a Pseudomonas fluorescens Migula infection experiment. Cells could be exposed to cigarette smoke with robots smoking, which may mimic human behavior and make it possible to directly study the effects of cigarette smoke on lung epithelial cells.
This technique could be used to explore a cigarette smoke on other cell taps in the context of a pulmonary infection. The described approach to be the basis for the developmental of effective therapies against a cigarette smoking dorsal lung injury and C0PD extra
