Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.
Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.
Hematopoietic malignancies directly arising from the hematopoietic stem cell compartment and uncontrolled, rapidly progressing, and severe immune-mediated disorders are often indications to perform allo-HCT1,2. However, although accounting for the occurrence of the prognostic beneficial graft-versus-tumor response, donor lymphocytes are frequently inducing and promoting an unwanted immune-mediated attack of healthy tissue components within the allo-HCT recipient, a process that is called graft-versus-host disease3. Manifestations in the gut, the so-called intestinal GvHD, represent the most dreaded complication of acute GvHD, severe forms of which are routinely associated with a high mortality1,2,4.
Overall, murine models of allo-HCT have emerged as invaluable tools to identify and study immune-mediated mechanisms underlying the pathogenesis of GvHD5. However, kinetic assessment of, for instance, beneficial effects of novel therapeutic interventions over time in live mice is routinely based on the determination of clinical GvHD scores6. While these scores are suitable to reflect, for instance, the overall disease burden (i.e., the systemic GvHD), clinical scores lack the sensitivity to reliably mirror organ-specific manifestations (e.g., in the gut). Hence, conclusions, for instance with respect to gut-protective effects of a given therapeutic intervention, that are based on these scoring systems usually fall short.
Despite major advances through the invention of novel whole-body imaging modalities in combination with the usage of either bioluminescent or fluorescent genetic mouse models7,8, methodologies to directly and specifically assess the intestinal manifestation of GvHD in live mice are lacking. Hence, the rationale behind the protocol of the endoscopic assessment of the intestinal GvHD phenotype described in the next section is to overcome this obstacle. Furthermore, the motivation is also to reduce experimental mice numbers since, so far, a detailed assessment of the cellular, morphological, and molecular characteristics (e.g., by histopathology or molecular biology) of intestinal GvHD manifestation has ultimately required the sacrifice of the experimental mouse.
Our institution has previously reported on the methodology of a mini-endoscopic assessment of colonic manifestations in the course of syngeneic colitis models9. In the protocol presented here, we have refined and adapted the colonoscopic scoring matrix for alloresponse-driven colitis in live mice with intestinal GvHD upon transplantation of alloreactive HCT and donor lymphocytes in an MHC class I fully mismatched setting. We identified four parameters suitable to reflect intestinal GvHD-related colonic lesions. Furthermore, we established a system that allows a fine-tuned grading of any single determinant, resulting in a new score that readily informs the reader about the severity of intestinal GvHD present in a given mouse at a given time point. Histopathological analyses confirmed that an endoscopic score above a certain threshold is reliably predicting moderate-to-high grade tissue inflammation. Hence, mini-endoscopic evaluation appears to represent a working substitute for the gold standard histopathology that routinely requires the sacrifice of the experimental mice. Importantly, this protocol can be applied at virtually any given time point and can be repeatedly used during the course of the disease10,11. Furthermore, in contrast to the usage of bioluminescence-dependent approaches, no labor-intense and time-consuming measures like intercrossing genetically modified mice are required and, hence, the methodology can be applied on virtually any mouse line of interest.
Taken together, given the detrimental clinical perspective of allo-HCT patients with severe intestinal GvHD, rapid scientific progress and more insight into the molecular mechanisms underlying the immune pathogenesis are urgently needed. Similarly, important, ethical considerations demand that the gain of knowledge should be achieved with the usage of the minimal number of experimental mice. Hence, both recognized claims on the research community exploring intestinal GvHD can be advanced by implementing serial mini-endoscopic evaluations of the colon in the experimental work chain to monitor and grade intestinal GvHD in live experimental mouse models, as described and validated in the protocol presented here.
The experimental methods described here have been approved by the government of Mittelfranken, Bavaria, Germany.
1. GvHD induction
2. Assessment of Systemic GvHD
NOTE: Perform this procedure in a semi-sterile surrounding in an experimental room licensed and equipped for handling live mice within the animal facility.
3. Assessment of Intestinal GvHD
The current protocol, describing the mini-endoscopic evaluation of intestinal GvHD-associated lesions of the distal colon, has been established and validated in mice previously subjected to the systemic induction of a severe acute GvHD model. In this study, we used an MHC class I fully mismatched model system in which BALB/c mice were lethally irradiated, followed by the transplantation of T-cell-depleted allogeneic bone marrow and by the administration of GvHD-inducing alloreactive C57BL/6 CD3+ T lymphocytes. T-cell-depleted bone marrow cells and splenic T cells for GvHD induction were purified by magnetic separation. The purity of these cell populations was determined by flow cytometry, and representative results of the purification process are displayed in Figure 1, showing a sufficient depletion of T cells from total bone marrow cells and a consistent enrichment of spleen-derived CD3+ T cells prior to their transfer to previously irradiated mice. The clinical course displayed in Figure 2 demonstrates the reproducibly robust induction of the clinical systemic GvHD phenotype upon allo-HCT in the presence (WT, in black) vs. absence (noT, grey) of alloreactive donor T lymphocytes. The scoring system previously reported by Cooke et al. represents a sum core in which six parameters are assessed and graded: body weight, posture, activity, skin and fur texture, and stool consistency6. The control mice experienced only one, early occurring peak of the clinical score, between days 5 and 10, that was similarly observed in T-cell-receiving mice and is, therefore, largely due to the irradiation-associated systemic inflammatory response. Regardless, clinical scores of donor T-cell-receiving mice started to rise earlier, showed a higher total peak, only transiently decreased after initially peaking, and then, essentially increased again, continuously, over the remaining period of the experiment. Overall, these results are in agreement with the interpretation that T-cell-receiving mice show stronger and more progressive signs of systemic GvHD compared to noT mice.
The donor T-lymphocytes-receiving allo-HCT mice showed various signs of acute organ-related and systemic GvHD manifestations, overall resulting in high sum scores, while the control mice lacked moderate to severe affections, especially at later time points. However, signs of intestinal GvHD are underrepresented in the systemic GvHD scoring system, where the only assessed gut-related parameter is stool consistency. As shown in Table 1, mini-endoscopically assessable criteria were defined, specifically intended for the precise description, scoring, and grading of intestinal GvHD-associated lesions, by adapting criteria previously reported for the evaluation of syngeneic colitis to the context of alloresponse-driven colitis9. Figure 3 displays typical examples for each individual criterion, illustrating the type and extent of intestinal GvHD-associated lesions, thereby visualizing the grading matrix applied during the mini-endoscopic evaluation of the distal colon of GvHD-prone mice upon transplantation of MHC class I fully mismatched donor lymphocytes.
Figure 4A shows that intestinal GvHD sum score results that are based on criteria defined in Table 1 and displayed in Figure 2 easily enable the experimenter to discriminate donor lymphocyte-receiving mice with severe signs of intestinal inflammation from control mice that are essentially devoid of GvHD. To validate the mini-endoscopically based grading system, histopathological studies were performed by using a previously reported microscopic grading system12. The data in Figure 4B confirm that the colon of mice severely affected by GvHD-related inflammation, as evidenced by high colonoscopic sum scores, display similarly strong histopathological signs of inflammation, given a histopathological sum score of ≥2 postmortem. In contrast, colon tissues of control mice display no (score 0) or, at most, mild (score 1) histopathological signs of inflammation, in line with the virtual absence of mini-endoscopically detectable signs of colitis. Furthermore, as shown in Figure 4C, D, correlation studies between mini-endoscopically and histopathologically assessed colitis activity and systemic GvHD scores were performed. Importantly, these studies demonstrated that mini-endoscopically determined sum scores of ≤3 reliably predict the absence of mid-to-higher grade (i.e., ≥2) intestinal GvHD-associated colonic inflammation scores obtained from histopathological grading. Finally, the severity of endoscopically assessed intestinal GvHD shows a correlation with the systemic GvHD activity.
Figure 1: Flow cytometric quality assessment of magnetically purified T-cell-depleted bone marrow cells and allogeneic splenic CD3+ T cells. (A) Depletion of CD90.2+ bone marrow cells achieved by magnetic separation, using a commercially available purification kit. The purity of T-cell-depleted bone marrow cells was determined by flow cytometry, by staining cells prior to and after T cell depletion with anti-CD45.1 and anti-CD3. Results from one representative experiment are shown. (B) Splenic T cells were magnetically purified, employing a commercially available purification kit. The purity was determined by flow cytometry, comparing the frequencies of CD45.2 and CD3 costaining of samples derived from before and after T cell isolation. Results from one representative experiment are shown. Please click here to view a larger version of this figure.
Figure 2: Clinical course of GvHD, employing an MHC class I fully mismatched model. To induce acute GvHD, BALB/c mice were whole-body irradiated on day 0. The mice received T-cell-depleted bone marrow cells 24 h later (d1). On day 2, the mice were injected with allogeneic splenic CD3+ T cells (wild-type [WT]; n = 9). As a control, some mice received T-cell-depleted bone marrow alone (noT; n = 9). The mice were specifically assessed three times a week for the presence and severity of clinical symptoms of GvHD. The displayed data represent mean values ± SEM of the clinical scores obtained from individual mice of the indicated treatment group from two representative experiments. The data were analyzed by two-way ANOVA, followed by Bonferroni's multiple comparisons posttest. ****p < 0.0001 was considered significant. Please click here to view a larger version of this figure.
Figure 3: Representative images illustrating grading examples underlying the scoring matrix of the intestinal GvHD-related colon alterations assessed by a mini-endoscopic evaluation of the colons of live, allo-HCT-treated mice. To complement and illustrate the detailed description of the used scoring grading and system in Table 1, representative mini-endoscopic images of the evaluated severity levels (grading) for all parameters individually in the colon of anesthetized, live mice undergoing allo-HCT between days 26 and 30 before as described in Figure 1 are displayed here. Please click here to view a larger version of this figure.
Figure 4: Mini-endoscopic scoring and grading of intestinal GvHD-related inflammation of the colon, reliably predicting the presence of higher-grade colitis assessed by histopathological scoring. (A) Between days 26 and 29 after the induction of GvHD as described in Figure 1, the manifestation of intestinal GvHD was evaluated by mini-endoscopy in live, anesthetized mice. Colonic alterations were scored and graded according to the scoring and grading system depicted in Table 1 and Figure 2. Representative endoscopic images of control (no T cell; noT) and wild-type (WT), T-cell-receiving mice and the mini-endoscopically assessed score (top) are shown. (B) The mice were sacrificed 12 h after the mini-endoscopic evaluation, and the distal part of the colon was processed and histopathologically assessed. The inflammatory activity of hematoxylin- and eosin-stained cross sections of the distal colon was graded. Representative hematoxylin- and eosin-stained cross sections of the distal colon of no T-cell- and WT T-cell-receiving allo-HCT-treated mice and the corresponding histology scores are shown. The data in panels A and B were analyzed by Student's t-test and are shown as mean ± SEM. ****p < 0.0001 was considered significant. WT: n = 15; noT: n = 15. The data represent pooled data from four individual experiments. (C) Interdependence of colonoscopy scoring and histological scoring. The data are shown as box plots. Each blot displays the median (black line within each box), the range of the data (min to max), and quartiles (areas of each box). WT: n = 15; noT: n = 15. The data represent pooled data from four individual experiments. (D) Correlation between colonoscopy scoring and clinical scoring. Both control and wild-type T-cell-receiving mice are included in the correlation analysis. The solid line represents the linear regression line. Spearman correlation coefficient (r-value) and p-value are shown. WT: n = 12; noT: n = 13. The data represent pooled data from three individual experiments. Please click here to view a larger version of this figure.
Score | 0 | 1 | 2 | 3 |
Translucency of the colonic wall | Extra-intestinal, inner organs (e.g. spleen) thoroughly visible | Discrete reduction of the visibility of extra-intestinal organs due to mild opacity of the colonic wall | Moderate reduction of the visibility of extra-intestinal organs due to significant opacity of the colonic wall | Lack of the visibility of extra-intestinal organs, i.e. non-transparent colonic wall |
Granularity | Smooth, unaffected mucosal surface appearance; colonic crypt pattern visible | Discrete roughening and cobblestone appearance of the mucosal surface | Moderate roughening and cobblestone appearance of the mucosal surface | Severe roughening and cobblestone appearance of the mucosal surface; cushion-like appearance of the mucosa |
Vascularity | Unaltered vascular pattern displaying the communicating network of large and small vessels | Discrete alterations of the vascular pattern; vessel pattern seems to fray | Some vessels are invisible; discontinuous vessel network | Contact induced bleeding; dot-like pattern of the vessels |
Stool consistency | Normal; stool is hard; fine threads of mucus between feces and colonic wall can be observed |
Stool is still shaped but may contain ragged edges; deformable with the tip of the endoscope | Stool is soft and unshaped stool is visibly more shining (higher water content) |
Stool is loose, liquid; watery diarrhea. Stool may be randomly spread over the mucosal surface of the colon |
Table 1: Mini-endoscopic scoring and grading matrix of intestinal GvHD-related lesions in the colon of live allo-HCT-treated mice. Alterations of the endoluminal and transmural colon morphology in allo-HCT pretreated mice were assessed by a colonoscopy of the rectum and the distal colon of anesthetized, live mice, using a murine mini-endoscopy system. Colonic lesions were classified with the help of the endoscopic scoring system (modified murine endoscopic index of colitis severity [MEICS]) that is deduced from the original MEICS scoring system reported by Becker et al.9 and adapted to the context of allo-HCT. The distal colon is visually assessed for the presence and magnitude of the following four parameters: 1. the transmittance of endoscopic light (translucency) through the colonic bowel wall as thickening of the wall; 2. the mucosal surface displaying a cobblestone appearance (granularity) of the endoluminal-oriented mucosal surface; 3. an altered vascularization pattern (vascularity); 4. endoscopically assessed stool appearance in situ (stool consistency). Each parameter is scored from 0 (no signs) to 3 (most severe phenotype), adding up to a maximum sum score of 12 per mouse.
The protocol describes the methodology of induction and mini-endoscopic assessment of the colonic phenotype observed in the course of intestinal GvHD. It serves the wider purpose of enabling scientists to study intestinal GvHD longitudinally and noninvasively over the entire disease course (i.e., from the onset of colonic manifestation and progression until maximal disease activity).
However, there are some critical steps and important limitations inherent to the presented methodologies the experimenter needs to be aware of prior to applying the technologies in the respective scientific context. First, although we have successfully used the mini-endoscopic assessment of intestinal GvHD-related colonic lesions in a minor histocompatibility-mismatched model system11, the provided protocol here is currently exclusively applicable to MHC class I fully mismatched acute GvHD models. While the MHC class I fully mismatched GvHD model is common and frequently used5, it is well established that the characteristics of GvHD manifestation are highly dependent on the used mouse substrains since this strongly impacts, for instance, the sensitivity to irradiation. Furthermore, the experimental mouse source with, for instance, a varying composition of intestinal microbiota and used numbers of transferred allogeneic T cells may critically impact the kinetics and dynamics of the intestinal GvHD manifestation12. Hence, a critical step is to carefully test and validate the indicated measures for their ability to successfully induce intestinal GvHD as shown.
Regarding the successful implementation of the live mini-endoscopic assessment of acute intestinal GvHD, we want to stress that, while handling the endoscope is straightforward, it may require some training, to minimize the risk of experiencing the most dreaded complication (i.e., to perforate the intestinal wall with the rigid instrument in combination with the need to air-inflate the colon). Due to an increased inflammation-induced vulnerability and decreased flexibility of the colonic tissue, the gut wall integrity might be more at risk during the postradiation recovery phase (i.e.,until day 10) and upon the full manifestation of intestinal GvHD-related colitis (i.e., after approximately day 25). Importantly, however, we have not observed significantly increased complication rates dependent on the time point of endoscopy, as long as the endoscope is gently advanced under sufficient visibility. In contrast, we identified suboptimal drug dosing to be a risk factor since an insufficient depth of anesthesia can result in the occurrence of unwanted body movements of the experimental mouse and, consecutively, colonic wall perforation events. Second, the most critical step during the scoring process represents restrictions in this matter due to the unwanted presence of solid or liquid (e.g., diarrhea) feces in the gut lumen as the experimenter moves the endoscope into the scoring position. In this situation, flushing the colorectal region with saline solution using a flexible plastic pipette is often required. However, since this procedure might compromise the scoring accuracy and specificity of several parameters (e.g., stool consistency), this caveat carefully needs to be taken into consideration upon scoring.
There are several limitations restricting the interpretation and conclusions drawn from this endoscopic approach. First, intrinsic to a methodology using an inflexible endoscope via the rectal route, evaluations are limited to the last 3-4 cm of the gastrointestinal tract (i.e., the rectum and the distal colon). Therefore, GvHD-associated affections of the proximal colon and even the small intestine are by default excluded from the evaluation, indicating that scientific questions focusing on the small intestinal phenotype of GvHD will not benefit from this approach. Second, although inflammation is one of the hallmark morphologic features of intestinal GvHD, additional characteristics, like an increased apoptosis rate of the intestinal epithelial cells and a loss of the intestinal crypt architecture, are often found and included in the histopathological workflow underlying intestinal GvHD scoring and grading by pathologists in allo-HCT patients. Here, we restrictedly correlated the mini-endoscopically assessed intestinal GvHD scores with histopathologically assessed signs of inflammation. Taking this approach, we came to the conclusion that colonic inflammation above a certain threshold in the colonoscopic scoring is reliably predicting the presence of moderate-to-high grade inflammation, as assessed by histopathological postmortem analyses. However, future studies need to investigate how, for instance, histopathologically assessed apoptosis rates relate to the mini-endoscopic total sum scores or to, for instance,one of the four individual parameters embedded in the sum score. Third, while mice suffering from histopathologically moderate-to-high-grade GvHD can be readily identified by the endoscopic scoring approach, mice with more subtle signs of intestinal inflammation may be missed by the described approach, given the fact that histopathological scoring revealed the presence of mild signs of inflammation in the group of noT mice undergoing allo-HCT alone, without the transplantation of alloreactive donor lymphocytes. Biologically, this finding might be feasible, since noT mice have been allotransplanted, and minimal colitis may, indeed, reflect the presence of low levels of GvHD due to an incomplete removal of T cells from the bone marrow cell fraction or the reconstitution of alloreactive T cells from the bone marrow over time. Future studies need to address these questions in more detail. Fourth, formally, this protocol focuses on fully established intestinal GvHD-associated colitis. However, as shown and published previously, the onset of colitis around day 15 can be endoscopically detected and quantitated11. Here, GvHD-prone mice could be clearly distinguished from noT mice in which the colonic phenotype might be solely due to residual signs of the irradiation-induced tissue damage or, rather, mirror the mucosal recovery response after irradiation11. However, future studies need to correlate endoscopic evaluations and aim at detailed histopathological analyses of GvHD-prone mice compared to control mice at earlier time points.
Finally, an interesting, helpful add-on application of the endoscopic scoring of intestinal GvHD is the usage of the endoscopic working channel to direct a minimized biopsy forceps to the anatomic structure of choice in the colon. This feature provides, as a sort of built-in-option, the possibility to take view-guided tissue biopsies that can be further analyzed by a series of downstream techniques like histopathology, immunofluorescence staining, and real-time PCR analyses of genes of interest. However, future studies need to formally assess whether, for instance, histopathological scoring and grading of endoscopically taken biopsies are equivalent to histopathological results based on conventionally taken samples (i.e., from euthanized mice).
Overall, with this protocol describing the induction and its mini-endoscopic evaluation of intestinal GvHD, we provide a methodology to assess, score, and grade intestinal GvHD in a relevant murine GvHD model system of allo-HCT. The endoscopic assessment can be repeated in the same mouse at multiple time points over time, eliminating the need to euthanize mice to sequentially assess intestinal GvHD-associated tissue pathology (e.g., in a therapeutic setting). The mini-endoscopic scoring results proved to be comparable to results obtained by the histopathological assessment of intestinal inflammation in the colon and correlated well with systemic GvHD activity. Additionally, this procedure can be combined with view-guided biopsies via the working channel of the endoscope. Ultimately, however, future studies have to assess how far histopathologically determined morphological features other than inflammation, that are frequently found in intestinal GvHD-affected lesions, relate to the results obtained with this mini-endoscopic assessment and scoring approach.
The authors have nothing to disclose.
This study was supported by the Collaborative Research Centers (CRC) 221 (CRC/TR221-DFG, #324392634; project B03) (to K.H.) and CRC 1181 (CRC-DFG, project B05) (to K.H.), both funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).
BIOBEAM 2000 Gamma Irradiator | Gamma-Service Meical GmbH | ||
Phosphate-buffered saline (PBS ) | Sigma-Aldrich Co. LLC. | D8662-6x500ML | |
Semken Forceps (lenght: 13 cm; serrated, curved tips) | Fine Science Tools | 11009-13 | |
Hardened Fine Scissors (lenght: 8,5 cm; straight tips; cutting edge: 24 mm) | Fine Science Tools | 14090-09 | |
RPMI-1640 Medium | Sigma-Aldrich Co. LLC. | R8758-500ML | |
Hypodermic needle (26G) | B. Braun Melsungen AG | 4657683 | |
1 mL syring | B. Braun Melsungen AG | 9166017V | |
50 mL tube | Sarstedt Ag & Co.KG | 62,547,254 | |
Cell strainer with a 40 µm mesh screen | BD Falcon | 352340 | |
Ammonium chloride (NH4Cl) | Sigma-Aldrich Co. LLC. | 11209 | ingredient of ACK lysing buffer |
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA) | Carl Roth GmbH & Co.KG | 8043.2 | ingredient of ACK lysing buffer |
Potassium bicarbonate (KHCO3) | Merck KGaA | 1,048,540,500 | ingredient of ACK lysing buffer |
CD90.2 MicroBeads, mouse | Miltenyi Biotec GmbH | 130-049-101 | magnet cell separation to isolate T cell-depleted bone marrow cells |
Pan T Cell Isolation Kit II, mouse | Miltenyi Biotec GmbH | 130-095-130 | magnet cell separation to isolate splenic T cells |
Alexa Fluor 700 anti-mouse CD45.2 Antibody (clone: 104; lot: B252126; RRID: AB_493731) | Biolegend | 109822 | |
Pacific Blue anti-mouse CD3 Antibody (clone: 17A2; lot: B227246; RRID: AB_493645) | Biolegend | 100214 | |
FITC anti-mouse CD4 Antibody (clone: GK1.5; lot: B225057; RRID: AB_312691) | Biolegend | 100406 | |
PE/Cy7 anti-mouse CD45.1 Antibody (clone: A20; lot: B217246; RRID: AB_1134168) | Biolegend | 110730 | |
APC/Cy7 anti-mouse CD8a Antibody (clone: 53-6.7, lot: B247008; RRID: AB_312753) | Biolegend | 100714 | |
Filtrated bovine serum | Pan Biotec | P40-47500 | ingredient of FACS buffer |
96-well polystyrene V-bottom plates | Greiner Bio-One | 651201 | |
Polystyrene Round-Bottom Tube (5 mL) | Falcon | 352052 | |
BD LSRFortessa II flow cytometer | BD Bioscience Co. | ||
Insulin syringe with sterile interior (30G) | BD | 324826 | |
Oxy Vet Oxymat 3 | Eickemeyer | oxygen concentrator for anesthesia | |
NarkoVet | Eickemeyer | 213062 | |
Plexiglass chamber | Eickemeyer | 214620 | |
Straight Forward Telescope | KARL STORZ SE &Co KG | 64301 AA | part of the experimental setup for colonoscopy |
Protection and Examination Sheath | KARL STORZ SE &Co KG | 61029 C | part of the experimental setup for colonoscopy |
Examination Sheath with working channel | KARL STORZ SE &Co KG | 61029 D | part of the experimental setup for colonoscopy |
Biopsy Forceps | KARL STORZ SE &Co KG | 61071 ZJ | part of the experimental setup for colonoscopy |
175 Watt SCB XenonLight Source | KARL STORZ SE &Co KG | 20132120 | part of the experimental setup for colonoscopy |
Fiber Optic Light Cable | KARL STORZ SE &Co KG | 495 NL | part of the experimental setup for colonoscopy |
Image 1 S3 Camera Head | KARL STORZ SE &Co KG | 22220030 | part of the experimental setup for colonoscopy |
Image 1 SCB Camera Control Unit | KARL STORZ SE &Co KG | 22200020 | part of the experimental setup for colonoscopy |
LCD monitor | Olympus | OEV181H | part of the experimental setup for colonoscopy |
Forane / Isofluran | AbbVie Inc. | B506 | |
Formaldehyde solution 37 % | Carl Roth GmbH & Co.KG | 7398.1 | |
5,0 mL Dispenser tip | Eppendorf AG | 30089456 |