This protocol is established to isolate and identify the group two innate lymphoid cells from the murine nasal mucosa and explore the certain effects and underlying mechanisms of the ILC two in nasal diseases. This technique enables the researchers to isolate ILC two from local mesothelial and expose the expression of surface antigen by flow cytometry. Our technique doesn't include complicated operations.
However, one important thing is trying to remove the flesh adhering to the little bones completely to avoid the impact of other nearby tissues Before starting the experiment, house wild type C57 black six male and female mice aged 8 to 12 weeks under specific pathogen free conditions provided with standard laboratory chow and water. To establish the allergic rhinitis model, emulsify 50 micrograms of ovalbumin in 0.2 milliliters of sterile PBS containing two milligrams of aluminum hydroxide and intraperitoneally inject 50 micrograms of emulsified ovalbumin into each mouse on zero, seven, and 14 days. On days 21, 22, 23, 24, and 25, intranasally instill mice with 50 micrograms of ovalbumin dissolved in 30 microliters of sterile PBS, followed by euthanizing the mice 24 hours after the last challenge.
Soak the head of the euthanized mouse up in 75%ethanol for five minutes and avoid ethanol entering the external nostril. Place the abdomen downward on the operating table. Cut off the four teeth and at the midline of the head make an incision and cut open the skin using scissors.
Then, remove the lower jaw, cut off the entire nose along the end of the palate, and place the tissue into a 60 millimeter Petri dish containing five liters of ice cold PBS. Using scissors and forceps, remove the flesh and muscles adhering to the bones. Next, transfer the mouse nose to a new 60 millimeter Petri dish containing five milliliters of ice cold PBS and wash the bones.
After the second wash, transfer the nose into a 1.5 milliliter micro centrifuge tube. Sufficiently smash the tissue and transfer it to a 15 milliliter tube containing two milliliters of prewarmed digest buffer. Fasten the lid and place the tube vertically in an orbital shaker at 37 degrees Celsius and 120 to 150 RPM for 40 minutes.
Next, add five milliliters of ice cold RPMI 1640 medium containing 10%fetal bovine serum to stop digestion. Then filter the content through a 70 micron cell strainer to remove solid fragments. After centrifuging the filtrate at 500 G for five minutes, gently discard the supernatant and resuspend the pellet in ice cold RPMI 1640 medium.
To repeat the centrifugation as demonstrated before, resuspend the cell pellet in four milliliters of 40%density gradient media. Next, gently insert a Pasteur pipet to the bottom of the tube to add 2.5 milliliters of 80%density gradient media. Centrifuge the tube at 400 G for 15 minutes at room temperature with the acceleration and deceleration rate set lower than the third gear.
Before draining the cells at the interface, remove the top layer of impurities to avoid potential contamination. Then use a pipet to drain the mononuclear cells'layer at the 40%by 80%density gradient media interface into a 15 milliliter tube containing two milliliters of ice cold. Wash the cells with ice cold PBS twice.
Harvest the cells and centrifuge them for five minutes at 500 G at four degrees Celsius. Resuspend the cell pellet in staining buffer and centrifuge again for five minutes at 500 G.Discard the supernatant. Resuspend and incubate the cells in 80 microliters blocking solution for 30 minutes at four degrees Celsius in the dark condition.
Add one microliter of fixable viability dye 520 right before adding 20 microliters of the surface staining antibody cocktail of the appropriate dilution to the sample. Also, set the matching isotype antibodies and fluorescence minus one as the negative controls. Next, wash the cells in 500 microliters of staining buffer by centrifuging at 500 times G for five minutes at four degrees Celsius.
Then resuspend the cell pellet in 200 microliters of staining buffer. Finally, add 50 microliters of vortexed absolute counting beads to the stained cells. Agitate and subject them to a flow cytometry analysis.
An ova induced murine model developed to explore the role of group two innate lymphoid cells showed that the frequency of nasal rubbing and sneezing in the allergic rhinitis mice was significantly higher than in the control group. In the nasal mucosa of healthy control mice, around 2 to 3%of lineage negative and CD 45 positive cells were identified. The absolute number of group two innate lymphoid cells varied from 2, 000 to 4, 000.
The flow cytometry analysis indicated a remarkable increase in the total number of group two innate lymphoid cells in allergic rhinitis nasal mucosa compared with that in healthy control mice. Higher expression of CD 226 was detected on the group two innate lymphoid cells in the allergic rhinitis mice than in the control mice. In addition, the mean fluorescence intensity of CD 226 was significantly up-regulated in the AR mice.
Be aware of the steps of removing the flesh adhering to the nasal bone, setting the acceleration and deceleration rate when centrifuging the media and the draining the mononuclear cells. The identified ILC twos can be a stand for flow cytometry characterization sorted for cell culture or stimulated in vitro, which would help explore the specific risk of ILC twos in nasal tissue. This procedure of isolating and characterizing lymphocyte, particularly in innate lymphatic cells in nasal mucosa, makes this protocol a preliminary reference for researchers exploring the local nasal immune environment.
