During the humoral immune response, B cells activated by antigen proliferate and secrete antibodies as the response progresses. The default I GM antibody ISOTYPE is substituted with the more diverse IgG iga or IgE isotypes to assess proliferation and class switching. In vitro splenic B cells are isolated from an experimental mouse using magnetic cell separation.
The cells are stained with the fluorescent dye CFSE to monitor proliferation, which is stimulated by treatment with bacterial L-P-S-L-P-S stimulation also induces class switching to the isotype IgG three, which along with proliferation, can be monitored by flow cytometry. Today I'll be showing you a technique for the isolation and activation of pure splenic B cells. This technique can be used to look at class switch recombination and proliferation in vitro, and it can also be used to answer key questions in the field of B cell immunity, including the function and regulation of activation induced citing in deaminase.
Begin by soaking a euthanized mouse with 70%ethanol. The mouse should be eight to 12 weeks of age To ensure full maturation of the immune system, use sterile scissors to make an incision through the skin and muscle of the left hypochondria of the abdomen. Use scissors or forceps to remove the spleen from the adjacent tissue.
Next, cut it into three large pieces in PBS. Then using the rubber end of a one milliliter syringe plunger, gently mash the spleen through a 70 micron cell strainer into PBS. Be sure to use a pushing motion rather than a grinding motion.
As grinding may lead to rupture of larger proliferating cells. Spin down the cells at no more than 350 G for five minutes. Then resuspend the pellet in PBS.
Count the resuspended cells using a hemo cytometer in a sterile five milliliter polystyrene tube. Prepare a suspension of one times 10 to the eighth cells per milliliter in PBS with 5%normal rat serum. A maximum of two milliliters can be added to each tube.
Next at 50 microliters of EP negative selection. Mouse B-cell enrichment cocktail for every milliliter of cells. Then cap the tubes and place them in the fridge for 15 minutes.
Add 100 microliters EP biotin selection cocktail for every milliliter of cells. Cap the tubes and place in the fridge for 15 minutes following the incubation with biotin selection cocktail mix EP magnetic nanoparticles and add 100 microliters for every milliliter of cells. Pipette the mixture, cap the tubes and place them in the fridge for five minutes.
Top the solution to 2.5 milliliters with PBS and place in an cept magnet For five minutes, invert the magnet in tube and pour quickly into a new polystyrene tube. Do not shake the tube since negatively selected cells will be magnetically bound to the tube walls. To further increase the purity of the cell suspension, place the new tube of cells into the magnet for five minutes, then pour again into a new tube.
This step may reduce overall cell recovery. B cell purity can be assessed by flow cytometric analysis of B-cell surface markers. Following enrichment, greater than 95%of the cells should be B two 20 positive.
Re suspended the isolated cells in warm PBS supplemented with 0.1%BSA at a final concentration of 1 million cells per milliliter from a five millimolar stock solution of CFSE dye diluted in sterile dimethyl sulf oxide. Add two microliters for every milliliter of cells in the tube. CFSE is used to monitor cell division of stimulated cells.
A process crucial for isotype switching. A final concentration of 10 micromolar is optimal for the staining of primary B cells. Incubated 37 degrees Celsius for 10 minutes in the dark.
Note that CFSE is light sensitive and should be kept in the dark when possible. Following the incubation, quench the stain by adding an equal volume of bovine calf serum to your cells and incubating on ice. For five minutes to wash the cells top up the tubes with culture medium at least five times the volume equivalent of media must be added to counteract the viscosity of the bovine calf serum and for the cells to produce a pellet then centrifuge at 350 G.Wash the cells twice more in culture medium, the cells are now ready for stimulation.
Prepare in vitro B-cell culture medium by supplementing RPMI 1640 with 10%fetal bovine serum and 50 micromolar Beto me capita ethanol. Next, prepare stimulation medium by adding LPS at a final concentration of 50 micrograms per milliliter. Aliquot 125 microliters of the stimulation medium into each well of a flat bottom 96 well tissue culture plate.
Prepare the CFS E stained B cells in culture medium without LPS at a final concentration of 3.2 times 10 to the six cells per milliliter. Add 125 microliters of the cell suspension to the wells containing your stimulation medium and mixed by pipetting gently. Each well now contains 400, 000 cells in a final LPS concentration of 25 micrograms per milliliter.
This provides optimal levels of cell proliferation and class switching. Although alterations to these values may be made as desired, incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 72 to 96 hours. After 48 hours, clusters of large proliferating B cells will be clearly visible under a microscope to look at class switching.
Remove the cells from the incubator and wash them twice in one milliliter of staining buffer to prevent non-specific antibody staining. Pellet the cells by spinning them down at 350 G.Then resuspend the cells in 100 microliters of staining buffer with 5%normal mouse serum and one microgram of FC block per million cells and incubate for 15 minutes on ice without washing. Add a fluorescently tagged anti-US IgG three antibody at one microgram per million cells incubate for 30 minutes on ice, wash the cells twice and resuspend them in two to 300 microliters of staining buffer.
The cells are now ready for assessment by flow cytometry. CFSE is optimally excited at 492 nanometers by an argon ion laser and emits at 517 nanometers. The excitation and emission spectrum of the IgG three antibody is dependent on its conjugate fluorescent dye.
Following magnetic enrichment, the cell suspension should look like a homogenous population of indistinguishable small circular cells. After 48 to 72 hours of stimulation, isolated clusters of enlarged proliferating cells will be clearly visible. A large portion of the non proliferating cells will appear small and granular as they undergo apoptosis.
CSR can normally be detected at the highest levels between 72 and 96 hours after stimulation before most of the cells begin to die. At this stage, the cells should have undergone a number of cell divisions with switched cells appearing in the later daughter cell population. Following these protocols, other techniques such as rna, AI mediated gene knockdown can be used to study the effect of any gene on the function of a ID and the process of class switch recombination.
