Examination Protocol for Immunization of B Lymphocyte Membrane Surfaces

Immunoglobulin (SmIg) is an antigen recognition receptor for B cells and a specific surface marker for B cells, which can be detected by direct immunofluorescence. By mixing fluorescein-labeled anti-Ig antibody with lymphocytes under certain conditions, the fluorescein-labeled anti-Ig antibody binds to the Ig on the surface of the B cells, and fluorescence can be seen on the membrane of the B cells under the fluorescence microscope. This method can be used to identify B lymphocytes.

• Mice are killed by cervical dislocation, and the spleens are dissected out and placed in a dish containing 6 ml of Hank's solution, ground with a 100-mesh steel mesh, and mixed well. The cells are washed again by centrifugation at 1000 rpm for 10 minutes. The supernatant is decanted and the deposited cells are restored to a volume of 1 ml, i.e., a cell suspension of about 1×10⁷/ml. Take another test tube, aspirate 0.4 ml of 1×10⁷/ml cell suspension, and add 3.6 ml of Hank's solution, i.e., 1×10⁶/ml cell suspension.
• Take two 2 ml centrifuge tubes, add 1 ml of 1×10⁶/ml cell suspension to each, centrifuge at 2000 rpm for 3 minutes in a tabletop centrifuge, and discard the supernatant. Add 100 μl of fluorescein-labeled rabbit anti-mouse Ig antibody to one tube, leave the other tube without antibody as control, and put it in the refrigerator at 4°C for 30 minutes.
• Remove the centrifuge tubes and wash the cells twice with Hank's solution to remove free antibodies. After the last centrifugation, discard the supernatant, leave a little reflux solution, mix well, drop the slides, and observe with a fluorescence microscope.
• Observed by fluorescence microscope, SmIg-positive cells can be seen ring or spot fluorescence under falling excitation light. The total number of lymphocytes in the same field of view is counted by transmitted light illumination with a tungsten lamp, and a total of 200-300 lymphocytes are counted, and the percentage of SmIg-positive cells is calculated.

Creative Bioarray Relevant Recommendations

• Creative Bioarray provides B lymphocytes from are isolated from peripheral blood. The method we use to isolate rabbit B lymphocytes was developed based on a combination of established and proprietary methods.

• Our SuperBeads® Human B Lymphocyte Isolation Kit isolates B lymphocytes from peripheral blood by immunomagnetic negative selection. The kit depletes other kinds of cells. The negatively isolated Human B lymphocytes are left in the sample and have not been touched with the SuperBeads.

Thoroughly remove free antibodies before titrating the film to avoid false positive results.

Animal Primary Cells

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