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Effect of permeabilization on peripheral blood lymphocytes of bovine leukemia virus (BLV)-infected cattle
H Ungar-Waron1, R Paz, J Brenner
1Department of Immunology, Kimron Veterinary Institute, Beit-Dagan, Israel.
Insights
Permeabilization enhances flow cytometry analysis of cell surface and internal proteins in bovine lymphocytes. This method revealed higher B-B2 molecule expression in bovine leukemia virus (BLV)-infected cattle, aiding in disease assessment.
Area of Science:
- Immunology
- Veterinary Medicine
- Cell Biology
Background:
- Cell surface proteins are crucial for lymphocyte subset identification using flow cytometry.
- Permeabilization allows detection of internal or partially expressed antigens.
- Bovine leukemia virus (BLV) infection impacts lymphocyte populations.
Purpose of the Study:
- To evaluate the effect of cell permeabilization on flow cytometry analysis of lymphocyte proteins in cattle.
- To assess differences in protein expression between BLV-infected and BLV-free cattle.
- To determine optimal methods for analyzing cell surface and cytoplasmic proteins.
Main Methods:
- Peripheral blood lymphocytes from BLV-infected and BLV-free cattle were analyzed.
- Cells were analyzed both intact and after permeabilization.
- Monoclonal antibodies (mAbs) targeting lymphocyte markers (BoCD4, BoCD8, B-B2) and heat shock protein 70 (Hsp70) were used.
- Fluorescence-activated cell sorting (FACS) was employed for analysis.
Main Results:
- BoCD4, BoCD8, and CD4 CD8-T-cell subsets showed consistent frequencies after permeabilization.
- The B-B2 lymphocyte molecule, partially expressed on intact cells, was more readily detected after permeabilization.
- Permeabilized cells showed significantly higher B-B2 staining in BLV-infected cattle compared to controls.
- Hsp70 expression increased similarly in both groups after permeabilization.
Conclusions:
- Flow cytometry analysis benefits from assessing both intact and permeabilized cells for comprehensive protein expression profiling.
- Permeabilization reveals B-B2 expression differences, potentially useful for diagnosing BLV infection.
- This approach improves the assessment of both cell surface and cytoplasmic protein expression in lymphocytes.
Abstract:
Cell surface proteins serve as markers for immunophenotypic characterization of lymphocyte subsets by appropriate monoclonal antibodies and fluorescence-activated cell sorter (FACS) analysis. By the same method, internal antigens or those that are only partially expressed on the cell surface can be determined after permeabilization of the cells. Peripheral blood lymphocytes obtained from bovine leukemia virus (BLV)-infected cattle and from BLV-free cattle were permeabilized and several lymphocyte populations were examined. BoCD4, BoCD8 and three CD4 CD8-T-cell subsets retained their original frequencies after permeabilization in both groups of animals. The recognition of the B-B2 lymphocyte molecule was only partially expressed on the cell surface of intact lymphocytes and was further revealed on permeabilization. The frequency of permeabilized, but not intact, cells stained with this mAb was significantly higher for BLV-infected cattle than for BLV-free animals (P = 0.006). Reactivities of an anti-heat shock protein (Hsp) 70 were measured before and after permeabilization of PBLs. Similar increased cell frequencies were obtained for both groups of bovines. These data indicate that flow cytometry studies should be conducted on both permeabilized and intact cells for a better assessment of protein expression on the cell surface, as well as in the cytoplasm.