M cells in the follicle-associated epithelium covering Peyer’s patch were detected by whole-mount immunostainig with anti Gp2 antibody. previously reported that glycoprotein 2 (Gp2), specifically expressed within the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for any subset of commensal and pathogenic enterobacteria, includingEscherichia coliandSalmonella entericaserovar Typhimurium (S.Typhimurium), by recognizing Cilostamide FimH, a component of type I pili within the bacterial outer membrane5. Here, we present a method for the application of a mouse Peyer’s patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously explained6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer’s patch was setup. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer’s patch were recognized by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer’s patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells. Keywords:Neuroscience, Issue 58, M cell, Peyer’s patch, bacteria, immunosurveillance, confocal microscopy, Glycoprotein 2 Download video stream. == Protocol == == 1. Preparation of bacterial cells == Streak glycerol stocked fluorescent bacteria (such as GFP expressingS.Typhimurium) on LB an agar plate containing 100 g/ml of ampicillin. Tradition a single colony from LB agar immediately in 2 ml of fresh LB medium. Add 0.5 ml of bacterial culture to 4.5 ml of new LB medium and incubate until optical density of 1 1.0 at 600 nm is definitely reached. Harvest bacterial cells by centrifugation (3,000 x g, 5 min, 4C). Discard the supernatant and wash twice with 5.0 ml of sterile phosphate buffer saline (PBS). Resuspend bacterial pellet with 5 ml of PBS, and use 50 l of the suspension containing approximately 107colony forming unit Cilostamide (CFU) as the inoculum. In case of using fluorescently labeled bacteria, the bacterial cells were labeled by fluorescence labeling reagent relating to standard protocol. == 2. Anesthesia == Fill a small plastic package (10 x 10 x 5 cm) with 5% (v/v) vaporized isoflurane mixed with air flow (flow rate: 200 ml/min). Anesthetize eight- to sixteen-weak-old male or female mice in the package. Move the mice to an NSD2 autopsy table after anesthesia. Continually anesthetize the mice by 2% (v/v) vaporized isoflurane mixed with air flow (flow rate: 200 ml/min) (Number 1). This is a terminal process. == 3. Ligated Peyer’s patch loop assay == Incise 1cm of the abdominal skin and then cut the abdominal peritoneum of an anesthetized mouse and take out the small intestine comprising the Peyer’s patch. Ligate the intestine with sewing yarn, taking care to avoid blood vessels. Notice: only bind one part of the intestine, and leave the other part loose. Inject 50 l of bacterial suspension or PBS (control) having a syringe into ligated Peyer’s patch loop within the loose part of the intestine (Number 2). Bind loose part, and close the mouse’s belly having Cilostamide a clip. After 1 h, remove the ligated Peyer’s patch loop, and euthanize the mouse by cervical dislocation. Excise Peyer’s patch from ligated Peyer’s patch loop. Flashing the apical part of Peyer’s patch by 1ml of PBS with using syringe attached to a needle to remove excess mucosal fluid and bacteria. Flashing the Peyer’s patch an additional two times. == 4. Whole mount staining and confocal microscopic analysis of Peyer’s patch == Fix Peyer’s patch in BD Cytofix/Cytoperm remedy on snow for 1 hr. Wash Peyer’s patch three times with 1ml of BD Perm/Wash buffer for 5 min, and then block with 1 ml of obstructing buffer comprising Cilostamide 0.1% (w/v) saponin, 0.2% (w/v) BSA in PBS for 30 min on snow. Add 200-collapse diluted anti-mouse GP2 monoclonal antibody Cilostamide (5 g/ml) to the specimen to detect M cells. Incubate for 2 hrs at space temperature or over night at 4C. Wash three times with 1 ml of chilly PBS, then add 200-collapse diluted anti-rat IgG antibody conjugated with DyLight549 (20 g/ml)to the specimen. Incubate sample on snow for.