R‐phycoerythrin‐conjugated antibodies are inappropriate for intracellular staining of murine plasma cells

Abstract

Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE-conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye-conjugated form of antibody has been rarely reported. In the present study, we discovered that PE-conjugated antibodies, but not FITC-or APC-antibodies, selectively stained lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP-PC-selective staining with PEantibodies was not due to interactions of antibody-epitope or antibody-Fc receptor. This unexpected staining by PE-antibody was not dependent on the mouse strain of LP-PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP-PCs were also selectively stained by PE-conjugated antibodies. Taken together, these results show that PE-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions

“…While it has been reported that ASCs non-specifically retain PE in the context of ICFC (Bohacova et al, 2021;Kim and Kim, 2013), our current study confirms and extends these findings to include PE/Cy7. A key finding shown here is that the mere act of repetitive centrifugation post-fixation appears to induce the ASC retention of PE.…”

“…PFA) and permeabilization (i.e. Sap) techniques, it has been reported that ASCs non-specifically retain PE-conjugated Abs thus making them unsuitable for ICFC (Kim and Kim, 2013). This was shown to be the case for ex vivo stained ASCs as well as those generated in vitro following B lymphocyte stimulation (Kim and Kim, 2013).…”

“…Sap) techniques, it has been reported that ASCs non-specifically retain PE-conjugated Abs thus making them unsuitable for ICFC (Kim and Kim, 2013). This was shown to be the case for ex vivo stained ASCs as well as those generated in vitro following B lymphocyte stimulation (Kim and Kim, 2013). To better characterize this phenomenon, we evaluated the ability of B220 +/-CD138 HI CD90.2 -IgD -ASCs (Figure 1A) from the SPL to retain a variety of non-specific isotype control Abs conjugated to allophycocyanin (APC), PE and PE/Cy7.…”

“…Within the field of flow cytometry, phycoerythrin (PE)-conjugated Abs are known to be extremely "bright" and provide large signal-to-noise ratios thus facilitating their use to detect rare protein targets (Batard et al, 2002). However, an under-appreciated study has previously shown that ASCs nonspecifically retain PE during ICFC (Kim and Kim, 2013). This artifact pertains to ex vivo stained ASCs and well as those derived in vitro.…”

R-Phycoerythrin Induces SGC-7901 Apoptosis by Arresting Cell Cycle at S Phase

“…R -PE is commonly used as a fluorescent label probe [ 6 , 7 , 8 , 9 ], as biological and clinical assays [ 10 ] and for in-flow cytometry [ 8 , 11 ]. It is also used as a pigment or natural food dye [ 12 ].…”