The overall goal of this procedure is to precisely isolate and process the mirin gingiva for further flow cytometry analysis. This is accomplished by first surgically isolating the gingiva in a meticulous manner. Next, the excised tissue is processed to generate a single cell suspension.
Then extracellular and intracellular staining with antibodies is performed. Finally, the sample is analyzed by flow cytometry. Ultimately, results can be obtained that show different cell populations in the gingiva and cellular changes occurring in the tissue following exposure to an immunological insult by flow cytometry analysis.
The main advantage of this technique, overing methods, like in OC chemistry or in mono fluorescence, is that it is a rapid and simple method to analyze large number of cells, allowing simultaneous analysis of several variables, as well as the sorting of various cell types for further experimentation. This method can help answer key questions in the immunology field, such as how microorganisms influence gingival homeostasis and how it relates to inflammation induced bone loss in an experimental model of periodontitis, We first had the idea for this method when we start to address the role of the RI cells during periodontitis. Visual demonstration of this method is critical as the gene GVA acceleration technique is difficult to learn because of the small size and the relatively inconvenient access to the MU one J gva.
To begin prepare media solutions and sterilized surgical instruments according to the text protocol. After euthanizing a mouse, according to your institution's animal care and use committee's approved methods, use sharp, blunt straight scissors to cut down both sides of the oral cavity, including the cheeks and the mandible ramus, and pull down the mandible directing standard scissors perpendicular to the plane of the palatial bone. Cut the soft and hard tissues in an imaginary line, one millimeter behind the third molars.
Next, make an incision within the soft and hard tissues, two millimeters behind the incisors. Then pull down the anterior part of the mouth to expose the nasal cavity with the scissors parallel to the palate. Place one bleed into the nasal cavity and use the other to make an incision at the vestibule cutting to the posterior incision.
Repeat the incision on the other side of the maxilla to detach it from the skull. Then cut it down the middle and trim the palatal tissue from each hemi maxilla to reach the alveolar bone using abs and forceps. Peel the gingival tissues from the anterior border and place it into a plate with PBS and 2%fetal calf serum or FCS to process the gingiva, transfer it to a culture dish with one milliliter PBS two milligrams per milliliter of collagenase type two and one milligram per milliliter of DNAs.
Type one with a number 15 sterile surgical blade. Mince the tissue well before transferring it to a 15 milliliter conical test tube. Wash any remaining tissue cells from the plate with one milliliter of P-B-S-F-C-S and transfer it to the tube.
Incubate the tissue in a preheated shaking incubator at 37 degrees Celsius and 200 RPM for 20 minutes. Then add 20 microliters of 0.5 molar EDTA and incubate for another 10 minutes. Next, add P-B-S-F-C-S up to 12 milliliters and centrifuge the sample at four degrees Celsius, 400 times G for eight minutes.
After removing the supernatant resus, suspend the cells two milliliters of P-B-S-F-C-S. Filter the sample through a 70 micron cell strainer after spinning the flow through at four degrees Celsius and 320 times G for five minutes. Resus suspend the cells in 300 microliters of P-B-S-F-C-S.
Finally, count the cells working under a hood with a cool temperature and the lights off in a total volume of 100 microliters, add 0.2 to 0.5 micrograms of each selected antibody per one times 10 to the sixth cells and vortex briefly. After incubating the samples at four degrees Celsius for 15 minutes in the dark, add two milliliters of P-B-S-F-C-S and centrifuge at 320 times G and four degrees Celsius for five minutes. To fix the cells, aspirate the supernatant, and then while vortexing resus, suspend them by adding dropwise 500 microliters of cold BD cyto perms.
Cyto effects incubate the tubes at four degrees Celsius in the dark for 40 minutes following the incubation vortex. Briefly then add one milliliter of BD perm wash, buffer, and spin at 800 times G at four degrees Celsius for seven minutes. After repeating the wash aspirate all except for 300 microliters of the wash buffer.
Then to stain the cells intracellularly in a total volume of 100 microliters, add one microgram each of selected antibody per one times 10 to the six cells. Vortex briefly and incubate in the dark for 30 minutes. After washing the cells twice as before and removing all the supernat resuspend cells in 300 microliters of fresh cold BD perm wash buffer.
Following a brief vortex, filter the suspension into fax tubes. Keep the tubes on ice or in the fridge in the dark until ready to carry out fax analysis. Gingival cells pooled from two naive mice.
Were run in an LSR two flow cytometer and analyzed using FlowJo software. The side scatter and forward scatter plots shown here demonstrate the distribution of cells. Gating strategy to identify lymphocytes, monocytes, dendritic cells, and granulocytes is indicated for comparison purposes.
This figure represents a fax plot illustrating gingival cells purified from P gingivalis infected mice 21 days after oral gavage in the setting of experimental periodontitis. An increase in the various immune cell populations can be easily detected in the infected mice as compared to naive mice shown here. Immune cells in the processed gingiva are identified by gating on cells expressing the hematopoietic marker CD 45.
These cells were further segregated into CD three positive T cells shown in this figure. Neutrophils were also identified according to the expression of the LY six G and CD 11 B molecules. Finally, Langer hand cells and epithelial subset of dendritic cells were identified by gating on MHC class two positive CD 11 C positive cells and expression of EPAM and RIN CD 2 0 7 Once mastered, this technique can be done in three and a half to five hours if it is done properly Following this procedure.
Other methods like real-time PCR can be performed in order to answer additional questions like single cell gene expression After its development. This technique paved the way for researchers in the field of immunology to explore the local response in the ging GVA against any insult in the field of periodontal research. This technique allowed us to elucidate the role of immune and non-immune cells of the G GVA in the pathogenesis of experimental periodontitis.
After watching this video, you should have a good understanding of how to isolate and process urine GVA in order to obtain a single cell suspension, which will be further subjected to an extra and intracellular vir staining for flow cytometry analysis.
