Immunology and Infection
Published: May 8th, 2016
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....
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)
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.......
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.......
|sterile mouse cage
|contains lid, cage bottom, and alpha-dri bedding
|autoclavable rodent chow (NIH-31M)
|RPMI medium 1640
|0.5 M EDTA (pH 8.0)
|fetal bovine serum (FBS)
|the concentration in the protocol is based on an activity level of 1.878 U/mg
|collagenase, type II
|the concentration in the protocol is based on an activity level of 245 U/mg
|feeding needle (18 G, 2" length)
|10 ml syringe
|Disposable Scalpel (15 blade)
|multi-purpose cups, 120 ml
|multi-position stir plate, 9-position
|stainless steel conical strainer, 3 inch
|1.5 ml tube
|100 μm cell strainer
|40 μm cell strainer
|50 mL conical tube
|1 ml syringe
|96-well plate, round-bottom
|anti-mouse CD16/32 (Fc block)
|(optional) fixable viability dye eFluor 780
|10% formalin, neutral buffered
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