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Cancer Research

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

Published: November 21st, 2018



1Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer's patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.

Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on TFH and GC B cell subsets.

The entire gastrointestinal tract from the beginning to the end is decked with an extensive lymphoid network that contains immune cells more than any other organ in human and mouse1. Peyer's patches (PPs) constitute a major component of the intestinal branch of this cellular immune organization, so-called gut-associated lymphoid tissue (GALT)2,3. Within PPs, thousands of millions of antigens derived from dietary materials, commensal microbiota and pathogens are being sampled continuously, and when necessary appropriate immune responses toward them are mounted thus maintaining intest....

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All studies and experiments described in this protocol were conducted under guidelines according to Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center.

1. Designing Experimental Set-up and Mouse Groups

  1. (Optional) Co-house the experimental mice to facilitate horizontal transmission of gut microbiota between experimental mice and to reduce non-specific variability within PP lymphocytes. Additionally, use littermate controls of the same gender .......

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In contrast to a previous protocol20, we have observed that PPs are not evenly distributed throughout the SI but are localized more densely towards the distal and proximal ends of the SI as shown in Figure 1A. Flow cytometric analysis showed that, if followed correctly, our protocol gives a PP lymphocyte population that demonstrates forward-side scatter distribution similar to splenocytes (

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Here, we describe a protocol optimized for flow cytometric characterization of TFH and GC B cells. One of the major advantages of our protocol is that it enables the isolation of up to 107 (average 4–5 x 106 cells) total PP cells from a single mouse (C57BL/6 strain) without any digestive process. We observed that the total cell yield was positively correlated with the number of PPs and could be estimated from the following simple equation which is helpful for experimental planning: "total .......

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We would like to thank Laura Strauss and Peter Sage for helpful discussions and support with flow cytometry analyses.


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Name Company Catalog Number Comments
anti-mouse CD4 antibody eBioscience, Biolegend* 17-0041-81 ,10054* For detailed information see Table 1
anti-mouse CD19 antibody eBioscience MA5-16536 For detailed information see Table 1
anti-mouse PD-1 antibody eBioscience 61-9985-82 For detailed information see Table 1
anti-mouse ICOS antibody eBioscience 12-9942-82 For detailed information see Table 1
anti-mouse GL7 antibody Biolegend 144610 For detailed information see Table 1
anti-mouse CXCR5 antibody Biolegend*, BD Bioscience 145512*, 551960 For detailed information see Table 1
anti-mouse BCL-6 antibody Biolegend 358512 For detailed information see Table 1
anti-mouse Foxp3 antibody eBioscience 17-5773-82 For detailed information see Table 1
Streptavidin-BV421 BD Bioscience 563259 For detailed information see Table 1
FixableViability Dye eBioscience L34957 For detailed information see Table 1
7AAD Biolegend 420404 For detailed information see Table 1
FcBlock (CD16/32) BD Bioscience 553141 For detailed information see Table 1
Collagenase II Worthington LS004176
Collagenase IV Worthington LS004188
Foxp3/Transcription Factor Staining Buffer Set eBioscience 00-5523-00
6-well,12-well & 96-well plates Falcon/Corning 353046,353043/3596
50 ml conical tubes Falcon 3520
40 µm cell strainer Falcon 352340
10 ml syringe-plunger Exel INT 26265
RPMI Corning 15-040-CV
PBS Corning 21-040-CM
FBS Atlanta Biologicals S11150
Orbital shaker VWR Model 200
Curved-end scissor
Fine Serrated Forceps
Small curved scissor

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