The overall goal of this impression cytology technique is to derive enough cells to immunologically phenotype the cells involved in ocular surface disease. This method can help answer questions in the field of ocular surface, such as which immune cells are important for ocular surface inflammation. The main advantages of this technique are that it is relatively non-invasive and simple to perform in the clinic and the lab.
The implications of this technique extend towards the therapy of ocular surface inflammatory disorders since it allows the identification of specific populations of immune cells. This method can provide insight into the ocular surface immune cell population. It can also be applied to the investigation of ocular toxicology and ocular fibrosis.
Generally, individuals new to this method will struggle because of a lack of experience in handling the low number of cells collected from the eye. After determining the participant's clinical status, apply one to two drops of topical anesthesia to the superior bulbar conjectiva and the inferior fornix of the participant's eyes. Wait four to five minutes for the stinging sensation of the anesthetic to wear off.
Then position an impression cytology device tangentially onto the conjectiva on the temporal bulbar side and gently press and hold the push button for two to three seconds. Using forceps, release the bulk harvested ocular membrane into a microcentrifuge tube containing one milliliter of culture medium followed by one minute of continuous scraping of the membrane with a 10 microliter pipette tip to recover the rest of the cells from the device. Be careful when scraping the membrane, as too much scraping will result in a combination of debris, whereas too little scraping can result in low cell use.
When the surface of the membrane becomes uneven, repeat a second collection from the same eye on the nasal side of the eye, as just demonstrated. Pull the cells with the first ocular sample suspension and collect two samples from the participant's other eye. Within two to three hours of collection, centrifuge the ocular cells and re-suspend the pellet in 50 microliters of Flow Cytometry Staining Buffer, containing phosphate buffered saline supplemented with 0.05%bovine serum albumin.
Next, stain cells with a panel of the antibodies of interest for 20 to 30 minutes at room temperature, protected from light. At the end of the incubation, wash the cells with one milliliter of staining buffer and re-suspend the pellet in 200 microliters of fresh staining buffer. Calibrate the Flow Cytometer channel voltages in the Cytometer setup panel and setup a tube of tracking beads to normalize the data acquisition according to the manufacturer's instructions.
Then, in the Flow Cytometry data collection software, click Setup QC and choose the correct Cytometer setup and tracking bead lot number. Load the beads and click the Start button. Now, re-suspend the cell suspension with gentle tapping and load the tube onto the machine.
Open the data collection software and click Preview. When Threshold Rate is stable, click Acquire. Monitor the sample level, clicking Stop when the sample runs out.
After acquiring 10, 000 events, generate a forward by side scatter area dot plot and use the polygon gate tool to draw a population one gate over all of the events with a forward scatter area value greater than five times 10 to the fourth to exclude the debris. Click Create Dot Plot to generate a new dot plot. Right click on the plot and choose Properties to open the Plot Editor window.
Choose P1 and click the forward scatter area on the x axis to change the axis displayed to CD3. Open a new dot plot for the P2 population and change the x axis to 7-AAD. Draw a gate around the 7-AAD negative population to select the viable CD3 positive cells.
Name this gate P3 and open a new dot plot. In the Plot Editor window, select CD3 for the x axis and CD4 for the y axis. Now use the P4 and P5 populations to open two new dot plots and set the x axes to display CD45RO positive cells and the y axes to display the CCR7 positive cells in each plot.
Then create a P6 gate around the CD3 positive, CD4 positive, CCR7 positive, CD45RO positive cells and P7, P8 and P9 gates around the CD3 positive, CD4 negative, CCR7 positive, CD45RO positive cells. CD45RO negative, CCR7 negative, CD4 negative, CD3 positive cells and CD45RO positive, CCR7 negative, CD4 negative, CD3 positive cells, respectively. The CD4 positive and CD4 negative populations can be characterized as CD45RO positive, CCR7 positive, central memory T cells and CD45RO positive, CCR7 negative effector memory T cells.
And CD45RO negative, CCR7 negative, T terminally differentiated effector memory according to their CCR7 and CD45RO marker expression. Of these subsets, the CD4 positive and CD8 positive effector memory T cells, are the major sub-population of T cells typically observed on the ocular surface of healthy humans. Once mastered, this technique can be completed within two hours, if performed properly.
While attempting this, other procedures such as ELISA, Western blot and other studies can be performed to answer additional questions on the cytokines and the genes involved in the signaling pathway. After its development, this technique paved the way for researchers in the field of ocular surface studies to evaluate the different immune cell types within a specific patient demographic of interest. After watching this video, you should have a good understanding of how to perform impression cytology and how to isolate and process immune cells from the ocular surface.
