Immunofluorescence analysis of Mouse anti-Human IgM (Heavy chain) Secondary Antibody was performed using log phaseRamos cells (IgM producing B-cell line). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with0.1% Triton™ X-100 for 15minutes, andblocked with 2% BSA for 1 hour at room temperature. The cells were labeled withMouse anti-Human IgM (Heavy chain) Secondary Antibody (Product # A18911) at 1:250 dilution in 0.1% BSA, incubated at 4degreecelsiusovernight and then with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) at a dilution of 1:2000 dilution for 45 minutes at room temperature (Panel a: green). Nuclei(Panel b: blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product #A30107, 1:2000 dilution). Panel d represents the merged image showing cytoplasmic (plasma membraneandgolgi-body like) localization. Panel e represents ARH-77 cells (IgM non-producing B-cell line) which is a negative model forIgM expression. Panel f represents control cells with no primary antibody to assess background. The images werecaptured at 40X magnification inCellInsightCX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) (DOI:10.1002/eji.1830100305; 10.3791/3573; 10.1016/0022-1759(94)00286-6; PMID: 566614).

Western blot was performed using Rabbit anti-Human IgM (Heavy chain) Secondary Antibody (Product # A18911) and an ~80 kDa band corresponding to Human IgM Heavy Chain was observed in Raji, Ramos and Daudi but not in IM-9, ARH-77, MOLT-4 and Jurkat8203. Whole cell extracts (30 µg) of IM-9 (Lane 1), ARH-77 (Lane 2), Raji (Lane 3), Ramos (Lane 4), Daudi (Lane 5), MOLT-4 (Lane 6) and Jurkat (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Product # A18911 (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP Conjugate (Product # A27036, 1:20,000 dilution) using the iBright FL1500 (Product # A44115). Chemiluminescentdetection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Raji, Ramos and Daudi are known to express IgM whereas IM-9 and ARH-77 express IgG and are negative for IgM. MOLT-4 and Jurkat, being T-cell lines, do not express immunoglobulins (DOI:10.1002/eji.1830100305; 10.3791/3573; 10.1016/0022-1759(94)00286-6; PMID: 566614).