This article describes a method for activating dendritic cells (DCs) under hypertensive conditions and their subsequent use in adoptive cell transfer. The process involves manipulating sodium and calcium ion concentrations to activate DCs, which are then injected into mice.
Begin with a multi-well plate containing a suspension of dendritic cells or DCs.
To mimic a hypertensive condition, add a culture medium containing a high concentration of sodium ions and incubate.
During incubation, sodium ions enter the cell, increasing the intracellular sodium concentration.
This triggers the exchange of intracellular sodium ions with extracellular calcium ions.
Increased internal calcium levels activate pathways that facilitate the generation of reactive aldehydes, which bind to the cellular proteins. This activates the DCs, which display these modified proteins on their surface.
Post-incubation, collect the cells in a tube and centrifuge.
Remove the supernatant. Add fresh buffer to resuspend the cells.
Draw the cell suspension into a syringe.
Take an anesthetized mouse and inject the cell suspension retroorbitally which is an area behind the eye socket.
This process of injecting activated immune cells is called adoptive cell transfer.
Pipette 900 microliters of the DC suspension into a 24-well flat flat-bottom falcon plate. Add 100 microliters of the high salt DC culture media to each well for a final sodium concentration of 190 millimolar. Label the plate, and place it in a humidified carbon dioxide incubator at 37 degrees Celsius for 48 hours.
After 48 hours, remove the plate from the incubator. Wipe out the cell culture media in a single well up and down to resuspend the CD11c-positive DCs. Transfer all of the suspension into a corresponding labeled 1.6-milliliter tube. Repeat this for each individual well that was plated.
Centrifuge all of the tubes at 300 times g and at four degrees Celsius for 10 minutes. Aspirate the supernatant, and resuspend the DC pellet with 100 microliters of sterile DPBS. Next, draw the DC solution from one of the tubes into a 1-milliliter syringe equipped with a 27-gauge half-inch needle.
Place the naive male, 10-week-old, C57 black six mouse under 2% isoflurane to achieve a stable surgical plane. Check the level of anesthesia with the lack of pedal reflex. Once a stable surgical plane is achieved, slowly and carefully introduce the needle into the retro-orbital space at an angle of approximately 30 degrees.
Slowly and smoothly, inject the CD11c-positive DC suspension. Once the injection is complete, carefully remove the needle for the retro-orbital space, making sure not to damage the eye. Then, remove the mice from the nose cone, and monitor them for approximately 30 minutes during recovery from anesthesia.
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