Detection of True IgE-expressing Mouse B Lineage Cells

The overall goal of the following experiment is to distinguish true IgE expressing B cells from B cells, expressing other IgE isotypes secreted IgE. During in vitro class switching assays binds to B cell surface proteins, which obscures the ability to measure class switching to IgE. The goal is achieved by first removing receptor bound IgE molecules prior to analysis by use of trypsin, which not specifically cleaves cell surface proteins while leaving endogenously produced intracellular immunoglobulin molecules intact.

Secondly, intracellular proteins are exposed by fixing the B cells with formin and perme the cells with methanol. Next, the cells are stained with fluorescently labeled antibodies to reveal intracellular immunoglobulins. The results show a distinct IgE positive B-cell population.

If trypsin is used based on flow cytometry analysis, Studies into IgE biology have been limited due to the difficulty to detect IgE producing cells. This method provides an accessible solution that laboratories can easily adopt to accelerate our understanding of this interesting molecule. This technique is advantageous over existing methods of eliminating the effect of receptor bound IgE.

For example, it is less costly than blocking surface IgE with anti IgE antibodies and more forgiving than an acid wash treatment. Though this method can provide insight into analyzing mouse B-cell class, switching to IgE treatment with trypsin prior to fact staining could also be applied to other protocols that require removal of surface proteins From a freshly harvested mouse spleen. Prepare a single cell suspension.

Gently press the organ through a 70 micron cell strainer using the plunger of a three milliliter syringe. Collect the cells in a 15 milliliter conical centrifuge tube containing 10 milliliters of one XPBS. Next centrifuge cells at 300 Gs for five minutes at four degrees Celsius.

Decant off the supra natant and resuspend the pellet in one milliliter of red blood cell lysing buffer. Let the lysing reaction go for five minutes before neutralizing it with 13.5 milliliters of one XPBS. Then centrifuge the cells again at 300 Gs for five minutes and resuspend the pellet in the residual volume.

Now add warm B-cell stimulation media containing IL four and anti CD 40 to the cells. Start with an eight milliliter culture divided into two wells of a six well culture plate and stimulate the cells at 37 degrees Celsius over five days. Check the culture daily to maintain the cells at 1 million per milliliter.

Adjust a concentration by splitting the cultures into other wells and diluting with B-cell stimulation. Media containing fresh IL four and anti CD 40. Begin with collecting up to 10 million of the stimulated B cells in a 15 milliliter conical tube.

Then centrifuge them at 300 Gs for five minutes and decant the supernatant to remove the residual protein from the cell collection media. Wash the B cells with 10 milliliters of PBS followed by another centrifugation and decantation of the snat After decanting, re suspend the cell pellet in the residual volume of about 100 microliters and carefully add 800 microliters of room temperature 0.1%trips in EDTA. Add the solution slowly along the sidewall of the tube.

Add an incline, then cap the tube and mix it gently running the liquid along the length of the tube. Five or six times a stringy white precipitate might form in the solution within one minute. Quench the reaction with one milliliter of cold FCS.

Keep the cells on ice. Now they are still alive and can express new surface proteins. Immediately dilute the FCS by adding 10 milliliters of cold PBS.

Remove any floating debris with a pipette. Spin down the cells at four degrees Celsius. Immediately decant the snat and re suspend the cells in the residual volume.

If necessary, bring the volume up to 100 microliters with cold PBS proceeding quickly in a fume hood at 800 microliters of 10%neutral buffered formin pipette the mixture up and down the tube will begin to warm up once mixed incubate the cells in the fixative for 10 minutes at 37 degrees Celsius. Meanwhile, set up a benchtop vortex mixer at a gentle shaking speed and prepare methanol cool to negative 20 degrees Celsius for use. When the cells are done incubating.

Draw nine milliliters of methanol into a serological pipette. Then uncapped gently vortex the tube while adding the methanol dropwise. After dispensing two milliliters of methanol dropwise speed up to a slow stream.

Now let the tube sit on ice for 30 minutes to perme the cells. Alternatively, the cells can be stored at negative 20 degrees Celsius for up to a month. Begin the staining procedure by first centrifuging the perme cells and decanting the methanol.

A white precipitate may pellet with the cells. Then wash the cells by mixing in 10 milliliters of room temperature one XPBS centrifuging and decanting. Perform this wash step twice.

Next, do an additional wash with 10 milliliters of 2%FCS fax, buffer and resuspend. The pellet in the residual volume proceed by loading the sample into one well of a 96 well round bottom plate. Many samples are typically processed in parallel.

Load 20 to 50 microliters using a plate spinner centrifuge the cells at 300 Gs for five minutes. Then decant the snat and put the plate on ice. Now stain the cells to each well add 60 microliters of 2%FCS fax buffer containing four different antibodies.

Let the cells incubate in the stain for five minutes after the stain. Wash the cells by adding 150 microliters of 2%FCS fax buffer fur. Then centrifuge the plate as before, followed by decanting the solution and resus suspending the cells.

Use 400 microliters of 2%FCS fax buffer. Now pipette the cells through a 40 micron nylon cell strainer into a five milliliter fax tube for the fax gate. The cells by first isolating CD 45 R positive cells, also known as B two 20 positive cells from the blasting lymphocyte population on the F-S-C-S-S-C plot then from the B two 20 positive cells.

Use the IgE and IgG one plot to reveal the corresponding populations using this procedure. Class switch recombination to IgE was studied in mouse B cells using a gating strategy based on the blasting lymphocytes. A clear population of IgE positive cells could not be cleanly discriminated without the trypsin of the cells trypsin prior to fixation and permeation enabled separation of the IgE producing cells due to the removal of surface bound IgE Once mastered.

This staining technique can be done in about an hour if it is performed properly. Working with formalin and methanol can be hazardous. Therefore, a fuma should always be utilized and proper personal protection equipment should always be used while performing this procedure.