The overall goal of this procedure is to quantify the level of neutrophil migration across a bladder epithelial barrier during bacterial infection, or in response to chemo attractant molecules. This is accomplished by first growing cultured bladder epithelial cells to confluence on permeable supports. The second step is to infect the bladder epithelial cells with various bacteria or apply chemo attractant molecules to the lower reservoir of the permeable supports.
Next neutrophils are isolated from venous blood and are applied to the upper reservoir of the permeable supports. The final step is to enumerate the number of neutrophils in the lower reservoir after one hour using a hemo cytometer. Ultimately, the trans uro epithelial neutrophil migration assay is used to assess differences in neutrophil migration elicited by various stimuli.
Though this method can provide insight into aspects of the innate immune response during urinary tract infection, it can also be used to investigate other diseases, including bacterial infections that occur at other mucosal surfaces. Perform this procedure in a tissue culture hood using a septic technique using sterile forceps. Invert the permeable supports in a sterile 25 millimeter deep tissue culture dish.
For optimal results, use 5 6 3 7 bladder epithelial cells that have undergone fewer than 10 subcultures trypsin eyes. A 75 square centimeter flask of 5 6 3 7 cells at 95%confluence according to the written text. Using a 1000 microliter pipette gently resuspend the cells in approximately two milliliters of RPMI plus to a concentration of 3 million cells per milliliter determined by cell counting.
Using a hemo cytometer apply 50 microliters of the cell suspension to each permeable support without touching the membrane. Place the lid on the dish and carefully place the dish at 37 degrees Celsius with 5%CO2 for no more than 16 hours. Using sterile forceps.
Write the permeable supports into a 24 well plate containing 0.6 milliliters of RPMI plus per. Well add 0.1 milliliters of RPMI plus to the upper reservoir of each permeable support before incubating the plate at 37 degrees Celsius with 5%CO2. Replace the medium every two days by aspirating the medium from the upper reservoir, followed by the lower reservoir.
Then apply fresh RPMI plus in the reverse order, adding 0.6 milliliters to the lower reservoir and then 0.1 milliliters to the upper reservoir. Seven days after seeding the permeable supports with 5 6 3 7 cells. Assess confluence of the cells by filling the upper reservoir with 0.35 milliliters of RPMI.
Plus the cells are sufficiently confluent when the medium does not equilibrate between the upper and lower reservoirs. Prepare the bacterial inoculum and collect blood from adult volunteers as described in the text protocol in a tissue culture hood. Use a 10 milliliter pipette to gently transfer the blood to a fresh sterile 50 milliliter conical tube.
Add an equivalent amount of 3%dextran in 0.9%Sodium chloride. Incubate the tube upright at room temperature for 20 minutes without disrupting the lower layer. Carefully aspirate the upper layer and transfer it to a new 50 milliliter conical tube.
Pellet the cells by centrifugation at 300 times G for 10 minutes following centrifugation, discard the supernatant. Resuspend the cell pellet in a volume of 0.9%sodium chloride equivalent to the starting volume of blood. Then layer 10 milliliters of density centrifugation solution under the cell suspension.
Preserving the interface between the two phases, centrifuge at 400 times G for 30 minutes with no break before discarding the supernatant to lice the remaining red blood cells. Resus, suspend the cell pellet in 10 milliliters of cold 0.2%sodium chloride after incubating for 30 seconds promptly add 10 milliliters of cold, 1.6%sodium chloride to restore isotonicity pellet the cells by centrifugation at 300 times G for six minutes and discard the supernatant. Repeat these steps until the cell pellet appears to be free of red blood cells.
Typically three rounds of lysis using a 1000 microliter pipette resus. Suspend the cell pellet, primarily polymorphonuclear leukocytes or PMN in warm RPMI minus to a concentration of 10 million cells per milliliter as determined by cell counting. Using a hemo cytometer, keep the cells at 37 degrees Celsius until use.
Typically pm n viability and purity are greater than 99%as assessed by trian blue exclusion and visualization of nuclear morphology after staining respectively. For the migration assay, use only permeable supports bearing confluent. 5 6 3 7 cell layers.
Eloqua one milliliter of warm RPMI minus per well. In a 24 well plate preparing three wells per permeable support aspirate the medium from the upper and lower reservoirs of the permeable supports using sterile forceps. Transfer the permeable supports to the 24 well plate wash the permeable supports three times by transferring the supports from well to well.
If a bacterial inoculum is to be used, invert the permeable supports in a sterile 25 millimeter deep tissue culture dish. For experiments with live or killed bacterial stimuli inoculate the apical side of each permeable support with 60 microliters of RPMI for a mock infection or with the prepared bacterial inoculum. Place the lid on the dish and incubate at 37 degrees Celsius with 5%CO2 for one hour.
Then add 0.6 milliliters of RPMI minus per well to a 24 well low attachment plate. Preparing one well per permeable support. Prepare three wells containing 0.5 milliliters of RPMI minus to enumerate PMN input.
Alternatively, if a chemo attractant is being used in place of bacteria, add the chemo attractant to 0.6 milliliters of RPMI minus in the prepared 24 well plate. The next step for both experiments is to write the permeable supports into the 24 well low attachment plate. Then add 0.1 milliliters of the prepared PMN to the upper reservoir of each permeable support.
Add 0.1 milliliters of PMN directly to the wells containing 0.5 milliliters of RPMI minus incubate at 37 degrees Celsius with 5%CO2 for one hour. Using a 1000 microliter pipette. Rinse the underside of the membrane of the permeable support with media from the lower reservoir using sterile forceps, gently scrape the membrane against the edge of the well.
To remove additional PMN from the apical side, collect the PMN by gently scraping the bottom of each well with the 1000 microliter pipette tip. And transferring the PMN suspensions to micro fuge tubes. Enumerate the PMN using a hemo cytometer to calculate the total number of PMN in the lower reservoir.
Multiply the number of PMN in one square millimeter by 6, 000. Data can be reported as a percentage of input PMN or as PMN numbers normalize to 10 to the sixth. Input PMN adherence to the protocol outlined in this video with attention to detail enables the enumeration of PMN migration in response to stimuli, including bacteria and chemo attractant substances infection with non-pathogenic e coli strain MG 1655.
Heat killed MG 1655 or a PEC mutant elicits significantly more pm n migration than mock infection or infection with the wild type pec strain UTI 89 a cystitis isolate. The addition of chemo attractants, FMLF or IL eight to the lower reservoir results in significantly more PM and migration than mock treatment. After watching this video, you should have a good understanding of how to assess neutrophil migration across an epithelial barrier in response to chemotactic stimuli.
