B. Lymphocyte membrane surface immunoglobulin examination assay

Summary

The test for the examination of immunoglobulins on the membrane surface of B-lymphocytes can be used to: detect the presence of B-lymphocytes.

Operation method

direct immunofluorescence assay

Principle

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.

Materials and Instruments

ICR Mouse
Fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse Ig antibody Hank's solution
Benchtop centrifuge Tubes Suction tubes Pipettes

Move

1. Mice were killed by cervical dislocation, and the spleens were dissected out and placed in a dish containing 6 ml of Hank's solution, grinded with a 100-mesh steel mesh, and mixed well. 1 ml of cell suspension was put into a test tube, filled with Hank's solution, centrifuged at 1000 rpm for 10 min, and the cells were washed once. The supernatant was decanted and the deposited cells were restored to a volume of 1 ml, i.e. a cell suspension of about ^1x107/ml. Take another tube and take 0.4 ml of ^1x107/ml cell suspension and add 3.6 ml of Hank's solution to make ^1x106/ml cell suspension. 2. Take a 2 ml centrifuge tube and add 3.6 ml of Hank's solution to make ^1x106/ml cell suspension.2. Take two 2 ml centrifuge tubes, add 1 ml of ^1x106/ml cell suspension to each, centrifuge at 2000 rpm for 3 minutes in a tabletop centrifuge, and discard the supernatant. Add 100 ul of fluorescein-labeled rabbit anti-mouse Ig antibody to one tube, and leave the other tube without antibody as control, and place it in the refrigerator at 4 ℃ for 30 minutes.
3. Remove the centrifuge tube, wash the cells twice with Hank's solution to remove free antibody, discard the supernatant after the last centrifugation, leave a little reflux solution, mix well, drop the slides, and observe with a fluorescence microscope.

Caveat

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

Common Problems

Results

Under fluorescence microscopy, SmIg-positive cells showed ring or spot fluorescence under falling excitation light. The total number of lymphocytes in the same field of view was counted by transmitted light illumination with a tungsten lamp, and a total of 200-300 lymphocytes were counted, and the percentage of SmIg-positive cells was calculated.

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