Flow Cytometry and Immunohistochemistry Analyses

Flow cytometry analyses were performed on single-cells suspensions from spleen, bone marrow, lymph nodes as previously described (20 (link)). Antibodies include: CD43(S7), CD5(53-7.3), CD138(281.2), CD21/35(7G6), CD23(B3B4), CD11b/Mac-1(M1/70) and IgK from BD Pharmingen (San Diego, CA); B220(RA3-6B2), CD19(eBio1D3), CD8a(53-6.7), CD4(L3T4), CD3ε(145-2C11) and TCRβ(H57-597) from eBioscience (San Diego, CA); Ter119 and Gr1(RB6-8C3) from BioLegend (San Diego, CA); IgM and IgD from Southern Biotech (Birmingham, AL) and PNA from Vector Labs (Burlingame, CA). Immunohistochemistry (IHC) staining was performed on formalin fixed, paraffin-embedded tissue sections as described previously using the following antibodies: B220, CD3, PAX5, BCL6, IRF4 and CD138 (21 (link)). Immunofluorescence on paraffin-embedded tissues was performed using antibodies against Ki67 (SP6, Abcam, Cambridge, MA), and B220 (RA-3B62, BD Pharmingen). Images were acquired with a Nikon Eclipse 80i microscope (Nikon, Melville, NY) and processed on NIS-Elements AR 3.10 software (Nikon, Melville, NY).

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Yamamoto K., Lee B.J., Li C., Dubois R.L., Hobeika E., Bhagat G, & Zha S. (2015). Early B-cell Specific Inactivation of ATM Synergizes with Ectopic CyclinD1 Expression to Promote Pre-germinal center B-cell Lymphomas in Mice. Leukemia, 29(6), 1414-1424.

Publication 2015

B220(RA3-6B2), TCRβ(H57-597) Ki67 (SP6, Nikon Eclipse 80i microscope NIS-Elements AR 3.10 software

Antibodies Ar elements Bcl6 Bone marrow Cd11b Cd138 Cd3ε Cd43 Cells Embedded paraffin Flow cytometry Formalin Immunofluorescence Irf4 Lymph nodes Mac 1 Microscope Pax5 Spleen Tcrβ Tissues Vector

Corresponding Organization :

Other organizations : Cancer Genetics (United States), Columbia University, University of Freiburg

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Variable analysis

independent variables

Tissue samples (spleen, bone marrow, lymph nodes)

dependent variables

Cell populations identified by flow cytometry (CD43, CD5, CD138, CD21/35, CD23, CD11b/Mac-1, IgK, B220, CD19, CD8a, CD4, CD3ε, TCRβ, Ter119, Gr1, IgM, IgD, PNA)
Immunohistochemistry staining patterns (B220, CD3, PAX5, BCL6, IRF4, CD138)
Immunofluorescence staining patterns (Ki67, B220)

control variables

Experimental protocols for flow cytometry and immunohistochemistry/immunofluorescence as previously described (20, 21)

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