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Immunology and Infection

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Published: May 8th, 2016

DOI:

10.3791/54114

1Division of Infectious Diseases, Department of Medicine, Boston Children’s Hospital

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines — which contain the largest number of immune cells of any organ in the body — are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer's patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

The principal task of the small intestine is often considered to be the digestion and absorption of nutrients1. While this metabolic function is clearly essential, the small intestine has an equally significant role in protecting the host from the continual barrage of environmental antigens found within the lumen2. The intestinal tract separates the outside world (e.g., luminal antigens) from the internal environment of the host with an epithelial layer that is only a single cell layer thick. As such, the small-intestinal immune system has the formidable task of balancing its threshold for reactivity, allowing foreign antigens from the d....

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All studies were conducted under strict review and guidelines according to the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School, which meets the veterinary standards set by the American Association for Laboratory Animal Science (AALAS).

1. Horizontal Transmission of Bacteria via Co-housing (Optional) 

  1. To minimize exogenous contamination (particularly if using gnotobiotic mice), practice aseptic technique while assembling sterile disposable cages, using food and water t.......

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Flow cytometric analysis of single cell suspensions of small-intestinal lymphocytes should yield a discrete population of cells that have similar forward and side scatter characteristics as splenocytes (Figures 1A and 1B). The lymphocytes may begin to die if the tissue is not maintained at 4 °C during the initial stages of the isolation, resulting in the lymphocyte population having a lower forward scatter and being more difficult to separate from other epithelial an.......

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We present a protocol for the isolation and flow cytometric characterization of small-intestinal lymphocytes, including the LPLs, IELs, and lymphocytes in the PPs. For those interested in evaluating how changes in the microbiota affect the small-intestinal immune system, we detail the straightforward steps involved in the horizontal transmission of organisms between mice harboring different microbiotas. Although this protocol focuses on the small intestine, the procedure is the same for analysis of the large intestine, w.......

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NKS is supported by NIH award K08 AI108690.

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Name Company Catalog Number Comments
Sterile Gloves Kimberly-Clark 555092
sterile mouse cage Innovive MS2-AD contains lid, cage bottom, and alpha-dri bedding
metal feeder Innovive M-FEED
water bottle Innovive M-WB-300
card holder Innovive CRD-HLD-H
autoclavable rodent chow (NIH-31M) Zeigler 4131207530
RPMI medium 1640 Gibco 11875-119
dithiothreitol (DTT) Sigma D5545-5G
0.5 M EDTA (pH 8.0) Ambion AM9262
fetal bovine serum (FBS) GemBio 100-510
dispase II Invitrogen 17105-041 the concentration in the protocol is based on an activity level of 1.878 U/mg
collagenase, type II  Invitrogen 17101-015 the concentration in the protocol is based on an activity level of 245 U/mg
dissecting scissors Roboz RS-5882
feeding needle (18 G, 2" length) Roboz FN-7905
10 ml syringe BD 305482
PBS Gibco 14190-250
Disposable Scalpel (15 blade) Miltex 4-415
curved forceps Roboz RS-5211
straight forceps Roboz RS-5132
multi-purpose cups, 120 ml VWR 89009-662
stir bar VWR 58949-062
multi-position stir plate, 9-position VWR 12621-048
stainless steel conical strainer, 3 inch  RSVP
1.5 ml tube Eppendorf 0030 125.150
100 μm cell strainer Falcon 08-771-19
40 μm cell strainer Falcon 08-771-1
50 mL conical tube Falcon 352098
1 ml syringe BD 309659
96-well plate, round-bottom Corning 3799
anti-mouse CD16/32 (Fc block) Biolegend 101320
(optional) fixable viability dye eFluor 780 eBiosciences 65-0865-18
10% formalin, neutral buffered Thermo Scientific 5725

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