Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.
The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD).
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease.
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.
The intestinal mucosa encompasses a single layer of columnar intestinal epithelial cells (IECs), underlying lamina propria immune cells and the muscularis mucosae. Besides its role in the absorption of nutrients, the intestinal epithelium is a physical barrier that protects the body interior from luminal commensal bacteria, pathogens, and dietary antigens. In addition, IECs and lamina propria immune cells coordinate the immune response inducing either tolerance or response depending on the context and stimuli. It has been reported that the disruption of the epithelial barrier can precede the onset of pathologic mucosal inflammation and contribute to inflammatory bowel disease (IBD) encompassing both ulcerative colitis and Crohn's disease1,2,3,4,5,6,7. Individuals with ulcerative colitis present excessive transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) forming crypt abscesses, a finding that has been associated with severity of disease8,9. Although compromised epithelial barrier function and excessive immune responses are hallmarks of IBD, there is a lack of experimental in vivo assays to perform quantitative assessments of intestinal permeability and immune cell recruitment into the intestinal mucosa.
The most common methods used to study intestinal epithelial permeability and PMN TEpM employ ex vivo chamber-based approaches using IEC monolayers cultured on semi-permeable porous membrane inserts10,11,12. The epithelial barrier integrity is monitored either by measurements of transepithelial electrical resistance (TEER) or the paracellular flux of the Fluorescein isothiocyanate (FITC)-labeled dextran from apical to basal compartment13,14,15. Similarly, PMN TEpM is typically studied in response to a chemoattractant that is added in the lower chamber16. PMN are placed in the upper chamber and after an incubation period, PMN that have migrated into the basal compartment are collected and quantified. While these methods are useful, easy to perform and very reproducible, they are obviously reductionist approaches and do not necessarily represent an accurate reflection of in vivo conditions.
In mice, a common assay to study intestinal paracellular permeability is by oral gavage of FITC-dextran and subsequent measurement of FITC-dextran appearance in the blood serum13,17. The disadvantage of this assay is that it represents an assessment of overall barrier integrity of the gastrointestinal tract rather than that of regional intestinal contributions. In addition, Evans blue is commonly used to evaluate vascular leakage in vivo18 and has also been employed to evaluate intestinal mucosal permeability in mouse and rat19,20,21. The quantification of Evans blue in the intestinal mucosa requires extraction from tissue employing incubation in formamide overnight. Therefore, the same tissue cannot be used to study intestinal epithelial permeability and neutrophil infiltration.
Here we highlight a simple protocol that reduces the number of animals needed to collect reproducible data on colonic mucosal permeability and leukocyte transepithelial migration in vivo. We, therefore, recommend the use of FITC-dextrans that are easily detectable in blood serum without compromising the integrity of intestinal loops which can be harvested for further analysis. Of note, the intestinal ligated loops have been used in various species (including mouse, rat, rabbit, calf) to study bacterial infection (such as Salmonella, Listeria monocytogenes and Escherichia coli)22,23,24,25 as well as intestinal permeability26; however, to the best of our knowledge there are no studies investigating mechanisms of PMN TEpM in specific regions in the intestine such as ileum or colon that are commonly involved in IBD.
Here we describe the mouse intestinal loop (iLoop) model that is a robust and reliable microsurgical in vivo method that employs a well-vascularized and exteriorized intestinal segment of either the ileum or proximal colon. The iLoop model is physiologically relevant and allows the assessment of intestinal barrier integrity and PMN TEpM on living mice under anesthesia. We demonstrate two applications: 1) quantification of serum levels of 4 kDa FITC-dextran after intraluminal administration in the iLoop 2) quantification of transmigrated PMN in the iLoop lumen after intraluminal injection of the potent chemottractant Leukotriene B4 (LTB4)27. Moreover, utilizing the iLoop model with Jam-a-null mice or mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-a fl/fl) compared to control mice, we are able to corroborate previous studies that have reported a major contribution for tight junction-associated protein JAM-A to intestinal permeability and neutrophil transmigration15,28,29,30,31.
The iLoop model is a highly functional and physiological method that can be used to corroborate in vitro assays. Furthermore, this is a versatile experimental model that allows the study of various reagents that can be injected into the loop lumen, including chemokines, cytokines, bacterial pathogens, toxins, antibodies and therapeutics.
All animal experiments were conducted in accordance with the guidelines and policies of the National Institutes of Health and approved by the Institutional Animal Care & Use Committee at the University of Michigan.
1. Preoperative preparation
NOTE: This method was generated employing adult mice from C57BL/6 genetic background, aged 8 – 12 weeks. All mice were kept under strict specific pathogen free conditions with ad libitum access to normal chow and water. Results were obtained using C57BL/6, Jam-a – null mice (Jam-a-/-) or mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-afl/fl) and littermate Jam-afl/fl controls as previously described30.
2. Generation of the ileal loop
3. Generation of the proximal colon loop (pcLoop)
NOTE: For details about mice that were used for the generation of the pcLoop, see the information provided at the beginning of the protocol section.
4. Quantitative assessment of intestinal permeability: 4 kDa FITC-dextran assay
5. Quantitative assessment of migrated PMN into the intestinal lumen after intraluminal stimulation with chemokines
NOTE: Very few PMN reside in the intestinal mucosa at the baseline level. Pretreatment of animals with pro-inflammatory cytokines results in an inflammatory environment that facilitates PMN recruitment from bloodstream into the intestinal mucosa.
A schematic representation of the ileal loop and pcLoop models is depicted in Figure 1 and Figure 2, respectively. The anatomical pictures display the critical steps of the procedure including exteriorization of the intestinal segment (Figure 1B and Figure 2B), identification of an appropriate location for ligations that allows minimal disturbance of blood supply (Figure 1C and Figure 2C) and cleaning followed by ligation of cut ends of the iLoop that can be filled with reagent solution (Figure 1D and Figure 2D). Importantly, the iLoop model preserves vital blood supply and allows physiological absorption of applied reagents such as FITC-dextrans or the potent PMN chemoattractant LTB4. At the end of the assay, the iLoop should be inflated (as seen in Figure 1D and Figure 2D) and display normal mucosal perfusion with bright-red mesenteric vessels. Depending on the assay, blood is collected to measure FITC-dextran in serum or iLoop luminal contents are processed for quantification of PMN TEpM prior to euthanizing the animal.
In order to verify the accuracy of the iLoop model for the assessment of intestinal permeability, a FITC-dextran pcLoop assay was performed to evaluate the role of TJ-associated protein JAM-A in the regulation of intestinal barrier function in vivo. Of note, it has been reported that JAM-A deficiency lead to increased epithelial intestinal permeability in vitro28 and after oral gavage in vivo29. Herein, using the pcLoop model, a 2.5-fold increase in 4 kDa FITC-dextran serum levels was quantified in Jam-a-null mice (Jam-a-/-) compared to controls (Jam-a+/+) (Figure 3A)30. Furthermore, similar results were obtained with mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-a fl/fl) compared to littermate controls (Jam-a fl/fl) (Figure 3B)30. Therefore, the pcLoop model was able to corroborate previous studies that have reported a positive contribution for JAM-A to the intestinal barrier function.
Then pcLoop model was employed to study PMN recruitment into the intestinal mucosa and subsequent TEpM in vivo. As shown in Figure 4A, the number of PMN in the luminal content of the pcLoop was quantified by flow cytometry analysis. PMN were defined as cells positive for each of the cell-surface makers CD45, CD11b and Ly6G36. Circulating white blood cells were used as positive control for gating strategy. As expected, the number of PMN present in a segment of proximal colon similar to the pcLoop was low under physiological conditions (Figure 4B). Pretreatment with pro-inflammatory cytokines TNFα and IFNγ prior to surgery resulted in augmented numbers of PMN recruited in the pcLoop lumen. The administration of the PMN chemoattractant LTB4 led to a dramatic increase in PMN counts supporting a LTB4-dependent PMN recruitment (Figure 4B). Immunohistochemical staining of PMN in the colonic mucosa corroborate the elevated recruitment of PMN following stimulation with cytokines and LTB4 when compared with cytokine treatment without LTB4 (Figure 4C)30. The pcLoop model was employed to study the contribution of JAM-A to PMN TEpM by using Villin-cre;Jam-a fl/fl mice. Loss of epithelial JAM-A led to a reduced number of transmigrated PMN in the colonic lumen compared to littermate controls (Figure 4D)30. These findings strongly support a role for JAM-A in facilitating PMN migration across the intestinal epithelium and provide complementary insights to studies that have reported the involvement of JAM-A in PMN migration across vascular endothelium in various models of inflammation31,37,38.
Figure 1: The ileal loop model. (A) Schematic overview of the ileal loop model. Median laparotomy is performed on mice under anesthesia and placed on a temperature-controlled surgery board. (B) Exteriorization of the caecum (*), ileum and mesentery. Two adequate sites for ligation are identified (1,2). (C) Isolate a segment of 4 cm length: the first ligature (1) is placed close to the ileo-caecal junction and a second ligature (2) is placed 4 cm away from the first ligature. (D) Two small incisions are made in the mesentery (1, 2) to create a 4 cm length ileal loop. After removal of luminal content and ligation of cut-ends, reagents such as fluorescent markers and chemoattractants can be injected into the lumen. The ileal loop is well vascularized (black arrowheads). Please click here to view a larger version of this figure.
Figure 2: The proximal colon loop model. (A) Schematic overview of the pcLoop model. Median laparotomy is performed on mice under anesthesia placed on a temperature-controlled surgery board. (B) Exteriorization of the caecum (*), proximal colon, mesocolon and ileum. Two adequate sites for ligation are identified (1,2). (C) The first ligature (1) is placed close to the caecum and a second ligature (2) is placed 2 cm more distal from the first ligature. (D) The pcLoop is exteriorized, cleaned of luminal content and inflated with reagents such as fluorescent markers and chemoattractants. The pcLoop is a well-vascularized 2 cm segment of proximal colon (black arrowheads indicate blood supply). Please click here to view a larger version of this figure.
Figure 3: JAM-A regulates intestinal permeability in vivo. (A) JAM-A deficiency (Jam-a-/-) led to increased colonic permeability to 4 kDa FITC-dextran. Jam-a-/- (13x animals; black dots) were compared with Jam-a+/+ controls (12x animals; white dots). 4 kDa FITC-dextran (1 mg/mL) in HBSS was injected into the pcLoop lumen. Fluorescence was measured in blood serum after a 120 min incubation period. Data are expressed as means ± SEM; n = 3 independent experiments. ****P < 0.0001; Mann-Whitney U test. (B) Increased colonic permeability to 4 kDa FITC-dextran in Villin-cre; Jam-afl/fl (18x animals, black dots) compared to controls (Jam-afl/fl, 12x animals, white dots). Data are means ± SEM; n = 4 independent experiments. ****P < 0.0001; Mann-Whitney U test. This figure has been modified from Flemming S, Luissint AC et al.30. Please click here to view a larger version of this figure.
Figure 4: JAM-A promotes LTB4-dependent recruitment of PMN into the lumen of the pcLoop. (A) Gating strategy to quantify PMN (CD45+, CD11b+, and Ly-6G/Gr1+ cells) in luminal content by flow cytometry with fluorescent counting beads. Leukocytes from blood samples were used as a positive control for the gating strategy. (B) Number of PMN recruited into the pcLoop lumen after cytokine (TNFα+IFNγ, 100ng each) treatment (10x animals; white dots) or after a combination of cytokines and 1 nM LTB4 (10x animals; black dots). Black squares represent the number of PMN at baseline as assessed in an intact colonic segment identical in length to the pcLoop that was not subjected to any surgery or treatment with proinflammatory cytokines and LTB4 (9x animals). Data are the mean ± SEM (n = 3 independent experiments), Kruskal-Wallis test with Dunn's multiple comparison test. *P < 0.05, ****P < 0.0001. (C) Immunohistochemical staining of PMN (anti-Ly6G/Gr1 antibody) in the epithelium of the pcLoop after treatment with cytokines alone (left panel, TNFα+IFNγ) or a combination of cytokines and LTB4 (right panel). The number of PMN recruited in the pcLoop is increased in the presence of LTB4 (black arrowheads). Scale bar: 100 µm. (D) Number of PMN recruited in the pcLoop lumen in Villin-cre; Jam-afl/fl mice (11x animals; black dots) compared to Jam-afl/fl mice (10x animals; white dots) in response to 1 nM LTB4. Data are means ± SEM; n = 3 independent experiments. *P < 0.05; 2-tailed Student's t test. This figure has been modified from Flemming S, Luissint AC et al.30. Please click here to view a larger version of this figure.
The mechanisms responsible for dysregulation of intestinal barrier function and immune cell recruitment under pathologic conditions such as IBD are incompletely understood. Here, we detail a robust in vivo murine model that employs a well-vascularized exteriorized intestinal segment of either ileum or proximal colon and allows for assessment of intestinal permeability, neutrophil migration studies as well as other applications.
The iLoop is a non-recovery surgery that is performed on live animals. Anesthesia must be continuously monitored over the course of the experiment and evaluation of depth of sedation is mandatory. The most critical steps include (1) the isolation of the iLoop, (2) ligation of cut ends and, (3) the inflation of the iLoop by intraluminal injection of reagent solution. In each of these steps, bleeding can occur, compromising the blood supply of the iLoop and affecting accuracy of the results. Of note, in rare instances of intraluminal bleeding during the PMN TEpM assay, the flow cytometry gating strategy presented here will help to distinguish transmigrated PMN from PMN originating directly from the bloodstream (non-migrated PMN). Transmigrated PMN collected in the iLoop lumen express high levels of surface marker CD11b10 compared to circulating PMN (Figure 4D).
Given that the iLoop allows quantitative analyses of intestinal permeability and migration of blood PMN into the intestinal lumen, it is important to standardize the size of the absorbing mucosal area and the blood supply. In order to ensure consistency between animals, it is essential that a correct length of intestinal segment is exteriorized. The iLoop should be 4 cm for the ileal loop and 2 cm for the pcLoop and be perfused by comparable blood supply. Inconsistency in these parameters will also result in unequal distension of the iLoop after intraluminal injection of reagents and augment variability inter and between experimental groups. Furthermore, to avoid over-distension of the iLoop, we recommend that no more than 250 µL of reagent solution be injected in the lumen for the ileal loop and 200 µL for the pcLoop, respectively.
There are some limitations inherent with the nature of the procedure. The iLoop is a non-recovery surgery that is performed on live animals. This is a technically challenging microsurgical method; however, personnel can acquire surgical skills through practice. The average duration of the surgery should be short (maximum 15 min). We recommend 120 min as an ideal incubation time for measuring intestinal permeability and 60 min for the PMN TEpM. Incubation times can be reduced, but extended timepoints might affect the overall inflammatory state of the animal under anesthesia. In addition, the protocol from the start of the surgical procedure to sample collection / analysis cannot be paused.
This iLoop model presents key advantages with respect to existing methods: (1) the iLoop is fully vascularized and is more physiologically relevant, (2) in contrast to the oral gavage method that assesses the overall gastrointestinal tract integrity and depends on gastrointestinal motility13, the iLoop allows to study the properties of specific localized areas in the intestine (terminal ileum or proximal colon) that are commonly involved in IBD, (3) the iLoop is the first in vivo model that allows the quantitative study of PMN TEpM into the gut lumen as well as other parts of the intestinal mucosa, including lamina propria and epithelial factions30,35. It is possible to employ high versus low molecular weight FITC-labeled dextrans (4 to 150 kDa) to evaluate both size selectivity and/or severity of epithelial barrier defects in knockout/knock-in mice or various experimental models including, but not limited to, intestinal inflammation. In addition FITC-labeled dextrans can be quantified in other organs such as the liver39 or as a novel approach for studies of the blood brain barrier providing insights into the role of intestinal permeability in gut-liver and gut-brain axes40,41,42. Furthermore, this method offers the possibility to perform two loops in parallel (ileal loop and pcLoop in the same animal) and instill two different fluorescent labeled probes for analyses of barrier properties in distinct areas in the intestine. Along similar lines, generation of two loops in parallel can be employed to specifically evaluate ileum versus colon for differences or similarities in recruitment of immune cells in response to the same reagent.
Here, by using the pcLoop with Jam-a-null mice or mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-a fl/fl), we corroborate findings from previous studies that have reported a positive role for the TJ-associated protein JAM-A in intestinal permeability and PMN TEpM. The applications of the iLoop can be expanded to various reagents including antibodies, microbial pathogens and therapeutic drugs30,34,35. Of note, we used LTB4 (336.5 Da) to model PMN TEpM given that it is a well-accepted potent and physiologic PMN chemoattractant and its ability to induce TEpM at low concentrations (1 nM) in the physiologic range. However, our loop model is adaptable to other relevant chemoattractants. We have reported the use of the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF) to induce significant recruitment of PMN into the colonic lumen30. fMLF (437.5 Da) is a lower affinity chemoattractant in mice which requires much higher concentrations to be effective (1μM). This model is adaptable for use of CXCL1/KC, another potent physiologic chemoattractant that we have successfully used, yet CXCL1/KC is expensive and a relatively large molecule (11 kDa) that is less efficient in crossing the epithelial barrier. We have also demonstrated that neutralizing antibodies against leukocyte-specific integrin CD11b/CD18 (αMβ2) that were injected into the loop lumen prior administration of chemoattractant LTB4 resulted in reduced PMN TEpM corroborating results from in vitro studies10,30,35. Furthermore, the pcLoop was recently employed to study the effect of PMN versus epithelial glycans in controlling the rate of PMN TEpM43. Reagents were injected into the pcLoop lumen prior administration of chemoattractant LTB4. Therefore, with its broad spectrum of applications, the iLoop can complement and confirm findings obtained via in vitro assays. Ligated intestinal loops have also been used by others to study bacterial infection (such as Salmonella, L. monocytogenes and E. coli), therefore we believe that the ease in adaptability of this iLoop model can be used for these studies as well.
Following treatment with pro-inflammatory immune mediators, the iLoop can be used as an acute model of intestinal inflammation. Furthermore, the iLoop may enable studies elucidating the link between increased intestinal permeability and immune cell recruitment after exposure to intraluminal pathogens or in chronic inflammatory experimental models. Of note, we have recently observed by employing the pcLoop model that in response to high dose of proinflammatory cytokines TNFα and IFNγ (1 mg of each) intestinal paracellular permeability to 4 kDa FITC-dextran resulted in enhanced PMN recruitment into the pcLoop lumen in response to LTB4 in comparison to low dose cytokines (100 ng of each)30. Interestingly, here we show that increased epithelial permeability secondary to Jam-a deficiency did not lead to enhanced PMN TEpM but diminished it. All together these results suggest that intestinal paracellular permeability affects the rate of PMN TEpM but the correlation is not direct and depends on factors such as the expression of adhesion molecules (similar to JAM-A) that play an important role in both epithelial barrier function and leukocyte migration16. Future studies are needed to investigate the fine tuning of immune cell responses by the intestinal epithelium, and contributions to pathologic mucosal inflammation such as inflammatory bowel disease.
In conclusion, the iLoop model provides a major improvement to existing approaches for the assessment of intestinal permeability and PMN TEpM in vivo that will significantly aid in understanding mechanisms underlying the regulation of intestinal inflammation and IBD.
The authors have nothing to disclose.
The authors thank Dr. Sven Flemming of the University of Wuerzburg for his contributions to the establishment of the proximal colon loop model, Sean Watson for the management of the mouse colonies and Chithra K. Muraleedharan for helping with the acquisition of the pictures of the iLoop model. This work was supported by the German Research Foundation/DFG (BO 5776/2-1) to KB, R01DK079392, R01DK072564, and R01DK061379 to C.A.P.
Equipment and Material | |||
BD Alcohol Swabs | BD | 326895 | |
BD PrecisionGlide Needle, 25G X 5/8" | BD | 305122 | |
BD PrecisionGlide Needle, 30G X 1/2" | BD | 305106 | |
BD 1ml Tuberculin Syringe Without Needle | BD | 309659 | |
15ml Centrifuge Tube | Corning | 14-959-53A | |
Corning 96-Well Solid Black Polystyrene Microplate | FisherScientific | 07-200-592 | |
Corning Non-treated Culture Dish, 10cm | MilliporeSigma | CLS430588 | |
Cotton Tip Applicator (cotton swab), 6", sterile | FisherScientific | 25806 2WC | |
Dynarex Cotton Filled Gauze Sponges, Non-Sterile, 2" x 2" | Medex | 3249-1 | |
EZ-7000 anesthesia vaporizer (Classic System, including heating units) | E-Z Systems | EZ-7000 | |
Falcon Centrifuge Tube 50ml | VWR | 21008-940 | |
Fisherbrand Colored Labeling Tape | FisherScientific | 15-901-10R | |
Halsey Needle Holder (needle holder) | FST | 12001-13 | |
Kimwipes, small (tissue wipe) | FisherScientific | 06-666 | |
1.7ml Microcentrifuge Tubes | Thomas Scientific | c2170 | |
Micro Tube 1.3ml Z (serum clot activator tube) | Sarstedt | 41.1501.105 | |
Moria Fine Scissors | FST | 14370-22 | |
5ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap (35 µm nylon mesh) | Falcon | 352235 | |
Puralube Vet Ointment, Sterile Ocular Lubricant | Dechra | 12920060 | |
Ring Forceps (blunt tissue forceps) | FST | 11103-09 | |
Roboz Surgical 4-0 Silk Black Braided, 100 YD | FisherScientific | NC9452680 | |
Semken Forceps (anatomical forceps) | FST | 1108-13 | |
Sofsilk Nonabsorbable Coated Black Suture Braided Silk Size 3-0, 18", Needle 19mm length 3/8 circle reverse cutting | HenrySchein | SS694 | |
Student Fine Forceps, Angled | FST | 91110-10 | |
10ml Syringe PP/PE without needle | Millipore Sigma | Z248029 | |
96 Well Cell Culture Plate | Corning | 3799 | |
Yellow Feeding Tubes for Rodents 20G x 30 mm | Instech | FTP-20-30 | |
Solutions and Buffers | |||
Accugene 0.5M EDTA | Lonza | 51201 | |
Ammonium-Chloride-Potassium (ACK) Lysing Buffer | BioWhittaker | 10-548E | |
Hanks' Balanced Salt Solution | Corning | 21-023-CV | |
Phosphate-Buffered Saline without Calcium and Magnesium | Corning | 21-040-CV | |
Reagents | |||
Alexa Fluor 647 Anti-Mouse Ly-6G Antibody (1A8) | BioLegend | 127610 | |
CD11b Monoclonal Antibody, PE, eBioscience (M1/70) | ThermoFisher | 12-0112-81 | |
CountBright Absolute Counting Beads | Invitrogen | C36950 | |
Dithiotreitol | FisherScientific | BP172-5 | |
Fetal Bovine Serum, heat inactivated | R&D Systems | 511550 | |
Fluorescein Isothiocyanate-Dextran, average molecular weight 4.000 | Sigma | 60842-46-8 | |
Isoflurane | Halocarbon | 12164-002-25 | |
Leukotriene B4 | Millipore Sigma | 71160-24-2 | |
PerCP Rat Anti-Mouse CD45 (30-F11) | BD Pharmingen | 557235 | |
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD FC Block) | BD Bioscience | 553142 | |
Recombinant Murine IFN-γ | Peprotech | 315-05 | |
Recombinant Murine TNF-α | Peprotech | 315-01A |