The overall goal of this procedure is to obtain a leukocyte enriched population of innate immune cells from the brain of mice acutely infected with thyer mirroring encephalomyelitis virus or TMEV. This is accomplished by first intracranial infecting mice with TMEV. Next, the brain is harvested and gently homogenized 24 hours after the infection.
The third step of the procedure is to then utilize a continuous perca and discontinuous fol gradient to selectively purify the leukocyte enriched cell layer. Ultimately, the phenotype of the brain infiltrating leukocyte population involved in the immune response to the acute infection of the brain can be characterized by flow cytometric analysis. Hi, my name is Dr.Charles Howe.
The method that we'll be demonstrating today can help answer key questions in the field of neuroimmunology. For example, what are the immune cells that infiltrate the brain during acute infection? Demonstrating the method today will be Reagan, la France.
Corey, a senior research technologist in my lab To elicit brain infiltrating leukocytes. Attach a 27 gauge needle to an automatic one milliliter Hamilton syringe and set the syringe to liver 10 microliters. Draw 10 microliters of DMEM infected with two times 10 to the fifth PFU of the Daniel strain of TMEV or 10 microliters of sham infected DMEM into the syringe with careful attention to air bubbles.
Anesthetize a five to six week old mouse and confirm sedation by toe pinch while under anesthesia. Prepare the mouse for injection by quickly locating the appropriate coordinates in the frontal cortex region on the skull part, the mouse fur with the needle point along both AEs as appropriate for injection to make the injection orient the needle perpendicularly to the skull and press the needle through the bone cover to an approximate three millimeter depth. To express the virally infected DMEM or sham infected DMEM into the brain, press the Hamilton syringe button down and wait two seconds for the media to enter the brain before withdrawing.
Carefully remove the needle and place the mouse in a clean, dry cage. The mice will awaken from the anesthesia quickly and resume normal function within one minute, 24 hours after intracranial viral or sham infection. And before isolation of the brain infiltrating leukocytes, fill a round bottom Oakridge centrifuge tube with 10 milliliters of RPMI 1649 milliliters of per call and one milliliter of 10 XPBS and store the tube at room temperature.
Bring the FI call pack plus to room temperature and fill a 15 milliliter conical tube with five milliliters of RPMI and place it on ICE at this time as well. After sacrificing the animal by modified isof fluorine overdose, quickly remove the brain and place it into the 15 milliliter conical tube. Containing the five milliliters of RPMI then transfer the brain and RPMI solution to a seven milliliter glass Pyrex brand, 10 brook tissue grinder and gently homogenize the brain with 10 to 15 strokes.
Next, pipette an additional five milliliters of RPMI into the homogenizer and pipette the solution up and down twice. Transfer the approximately 10 milliliters of brain homogenate to the previously prepared round bottom ridge centrifuge tube and gently invert the tube two to three times to mix. Now spin the brain homogenate at 7, 800 Gs for 30 minutes at room temperature in a fixed angle rotor centrifuge.
After centrifugation, remove the myelin debris that has floated to the top of the gradient. The leukocyte layer floats above the red blood cell pellet. Strain the leukocyte layer through a 40 micrometer cell strainer into a 50 milliliter conical tube.
Bring the solution up to a 50 milliliter volume. With our PMI spin the tube containing the diluted cell suspension in a clinical centrifuge at 600 Gs for five minutes. At room temperature, aspirate the supernatant and collect the cell pellet.
Re suspend the pellet in one milliliter of fax buffer and add the cell suspension to a five milliliter round bottom fax tube. Carefully underlay the suspensions with one milliliter of the room. Temperature fi call pack plus spin the fax tubes at 800 Gs in the clinical centrifuge for 25 minutes.
At room temperature with no break, remove the white fluffy layer at the interface with a one milliliter pipette and transfer the layer to a new five milliliter fax tube. Wash the cells with four milliliters of fax buffer. Now spin the tube again in the clinical centrifuge to pellet the cells.
Then aspirate the super named and collect the cell pellet. Resuspend the leukocyte pellet in blocking buffer, and keep on ice. Then count the cells.
After isolating and counting the brain infiltrating leukocytes. Plate the cells in a V bottom plate in preparation for flow cytometric immunophenotyping. Then spin the cells in a clinical centrifuge.
Re suspend the pellet in 200 microliters of blocking buffer and incubate the cells for 30 minutes on ice. Next, add antibodies at a concentration of one to 200, and incubate the cells for another 30 minutes on ice this time in the dark. Then spin the stained cells in the clinical centrifuge and then aspirate the supernatant.
Add 200 microliters of fax buffer to the pellet and gently pipette up and down three times. Then wash the cells in the centrifuge two more times in the same way after the final wash, resus suspend the cells in a final concentration of 2%para formaldehyde and transfer the stain cells to fax tubes. Finally, submit the fixed cells for flow cytometry and analyze the files offline using a fax analysis program.
Mouse leukocytes were isolated from the brain by differential density, centrifugation of tissue homogenous prepared from animals at 24 hours after intracranial infection. As seen in these two dot plots LY six CG positive F four 80 positive CD 11 B positive inflammatory monocytes were found almost exclusively in the CD 45 high population consistent with the infiltration of these cells from the periphery. In contrast, LY six CG negative F four 80, low CD 11 B positive macrophages were found almost exclusively in the CD 45, low left two panels and CD 45 negative far right panel populations consistent with a resident phenotype.
After watching this video, you should have a good understanding of how to prepare leukocytes from the brain of acutely infected animals.
