Flow cytometry analysis of transcription factors in T lymphocytes

Abstract

Detection of transcription factors in immune cell populations, particularly in subpopulations that are represented at low frequencies in lymphoid and nonlymphoid organs, presents a particular challenge when using traditional methods such as western blot analysis. Therefore, development of flow cytometry-based methods which allow identification of transcription factors in specific immune cell populations is of main interest. Here we developed and optimized a methodology for rapid and convenient detection of the transcription factor BCL11B in T lymphocyte subpopulations using flow cytometry. The optimal protocol employs saponin and Tween 20 both during the fixation and permeabilization steps, and we demonstrate that it is efficient for three anti-BCL11B antibodies covering distinctive BCL11B epitopes. In addition, we prove that the method preserves the staining of surface markers.

Figure 1

BCL11B transcription factor is localized in the nucleus of CD4⁺ T lymphocytes. (A) Prior to immunocytochemistry and western blot analysis CD4⁺ T lymphocytes were isolated from spleen and lymph nodes by MACS technology, using anti-CD4 antibody-coated magnetic microbeads and magnetic column separation, as described in Material and Methods. The degree of purification was evaluated by staining an aliquot of the purified CD4⁺ T lymphocytes with anti-CD4-FITC-labeled antibodies and analyzed by flow cytometry. Purity of the separated CD4+ T lymphocytes was 92.4%. (B) Left: immunocytochemistry staining of cells cytospun on coverslips with anti-BCL11B antibodies (Clone BL1801) or prebleed rabbit serum, followed by FiTC-conjugated secondary antibodies, as described in Material and Methods. Middle: Hoechst counterstained of the cells shown in the left; right: Overlay. Images were obtained on an Olympus BX61 Microscope with 100 X objectives. (C) Left: Western blot analysis of cytoplasmic and nuclear extracts from CD4⁺ T lymphocytes purified as described in A. Right: Western blot analysis of nuclear extracts from 0.5×10⁶ (left lane or 1.5×10⁶ CD4⁺ T lymphocytes (right lane). Actin and HDAC2 were used as loading controls.

Figure 2

Detection of BCL11B by Flow Cytometry. Total splenocytes were surface stained with anti-CD4-FITC and anti-CD8-PE-Cy7 antibodies as described in detail in Material and Methods. This staining was followed by several combinations of fixation/permeabilization and permeabilization treatments, as follows: [Table: see text] Anti-mouse (clone BL 1801) was used followed by secondary Alexa 647-conjugated goat anti-rabbit secondary antibodies. Histograms depict BCL11B expression in gated CD4⁺ (solid black line), CD8⁺ (dotted black line), and CD4⁻CD8⁻ (solid gray line) cells. Secondary antibody (gray shaded). Comparison of the five methods used indicates that method # 2 allowed BCL11B detection with low background staining in CD4⁻CD8⁻ cells.

Figure 3

Three out of the four anti-BCL11B antibody tested clones recognize nuclear epitopes exposed through the optimized method of staining. Total splenocytes were surface stained with anti-CD4-FITC and anti-CD8-PE-Cy7 antibodies, fixed and permeabilized using Method #2 (Fig. 2) further optimized by the addition of 10 μg/ml RNAse in the permeabilization buffer. Anti-BCL11B antibodies, clones BL1801, BL1800, BL1799, and BL1798 were used for BCL11B detection followed by Alexa-647-conjugated goat anti-rabbit secondary antibodies. The histograms show that the staining with BL1800 and BL1801 anti-BCL11B antibodies on gated CD4⁺ (solid black lines), and CD8⁺ (dotted black lines), T lymphocytes resulted in optimal detection of BCl11B with minimal staining on CD4⁻CD8⁻ cells (solid gray line). BL1798 clone was less optimal in detecting BCL11B expression in CD8⁺, compared to CD4⁺ T lymphocytes, and also gave a higher background on CD4⁻CD8⁻ cells. This clone requires further optimization. Clone BL1799 did not detect BCL11B in any of the T lymphocytes subpopulations tested. Therefore, BL1799 is not recommended for detection of BCL11B by flow cytometry. Secondary antibody (gray shaded).

Figure 4

Staining for the surface markers CD4 and CD8 is preserved following fixation and permeabilization with optimized Method #2. Total splenocytes were surface stained with anti-CD4-FITC and anti-CD8-PE-Cy7 antibodies as described in detail in Material and Methods, followed by Method #2 of fixation and permeabilization and staining with anti-BCL11B antibodies. The dot plot graph depicts the percentages of CD4⁺ and CD8⁺ T lymphocytes in the spleen.