We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.
The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.
Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation3. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD4. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate3 but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.
Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD9 and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely10. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways11. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.
It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction14. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis15. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.
All the experimentation using murine tissues described herein was approved by the Institutional Review Board at the University of Pittsburgh and conducted in accordance with the guidelines set forth by the Animal Research and Care Committee at the University of Pittsburgh and UPMC.
1. Preparation for culture
NOTE: All the reagents are listed in the Table of Materials section and all solution compositions can be found in the solution composition table (Table 1).
2. Crypt isolation from murine colonic tissue
NOTE: Transfer the tissue on ice. Take the appropriate amount of basement membrane matrix out of −20°C storage, and thaw on ice. Each 24-well is plated with 15 µL of basement membrane matrix. Prepare CIB1, CIB2, and complete colonoid growth medium as described in section 1.
3. Passaging the colonoids
NOTE: Each well can generally be passaged 1:4 to 1:6 according to the density of the original well. Take the appropriate amount of basement membrane matrix out of −20°C, and place it on ice to thaw. Colonoids can be used for experiments after two passages. When passaging colonoids, the steps are performed in a biological safety cabinet to prevent contamination.
4. Terminally differentiating the colonoid cells
5. Inducing inflammation in differentiated colonoids with inflammatory mediators
6. Intestinal epithelial monolayers derived from established murine colonoids
NOTE: Murine intestinal epithelial monolayers are derived from murine colonoids that have been passaged a minimum of two times. To allow for successful monolayer formation in 3-5 days, it is imperative not to separate the colonoids into single cells. Fragmented organoids that have been enzymatically dissociated into cellular clusters are ideal for growth.
7. Measuring the net resistance of the epithelial monolayers using a voltohmmeter on days 3 – 5 of monolayer culture
NOTE: The chopstick electrodes resemble forceps and are asymmetrical in length. The longer arm of the probe is the basolateral electrode, and the shorter arm is the apical electrode. The probe can be difficult to insert between the interior and exterior of the cell culture insert. Placing the probe at a slight angle upon insertion, followed by vertical straightening of the probe, will prevent the probe from becoming stuck. Make sure to read the net resistance of each cell culture insert at a similar angle, as this can impact the values.
8. Calculating the TEER using net resistance measurements from the voltohmmeter
NOTE: Successful monolayer formation of the colonoids will give TEER measurements greater than 115 Ω·cm2.
9. Inducing inflammation in the epithelial monolayers with inflammatory mediators
The 3D intestinal colonoid culture system is an invaluable tool to study the intrinsic contribution of the epithelium to intestinal mucosal homeostasis. The described protocol provides detailed instructions on how to isolate crypts from C57BL/6J (WT) mice at 8 weeks of age and establish a long-term colonoid culture system that can be manipulated for multiple downstream applications. Upon the isolation and plating of crypts in the basement membrane matrix, the crypts appear dense and multicellular in structure when visualized by bright-field microscopy. They can be spherical in shape or elongated and cylindrical, resembling the single, crypt-like structure that is normally observed within the in vivo intestinal architecture (Figure 1A). By Day 1, the majority of the crypts form a compact, spherical structure, with a few colonoids starting to develop an interior lumen (empty space) devoid of epithelial cells (Figure 1B). As they continue to grow over the next several days, the diameters of the colonoids continue to increase, and the lumens of the colonoids become more defined (Figure 1C). By Day 5, each colonoid is approximately 100-250 µm in diameter, forming a large, prominent lumen surrounded by a thin layer of epithelial stem cells (Figure 1D). At this point, the colonoids are both enzymatically and mechanically disrupted for passaging. After two passages, they can be used for downstream experimentation.
To assess if the metabolic changes observed in the colonic scrapings of DSS-treated mice are retained in colonoids derived from mice on the same DSS regime, 8-week-old WT mice were treated with 2% DSS in drinking water ad libitum. Age-matched and gender-matched WT control animals were given drinking water ad libitum without DSS. After 7 days, the mice were sacrificed, and crypts were isolated from the colons of both the control (n = 3, male) and DSS-treated animals (n = 3, male) to establish colonoids. After two passages, protein was isolated from each set of colonoids, and the expression of peroxisome proliferator-activator receptor-gamma coactivator-1α (PGC1α), the master regulator protein of mitochondrial biogenesis, was assessed via western blot analysis (Supplementary Figure 1). There was no significant difference in PGC1α expression when comparing the colonoids established from the DSS-treated mice versus the colonoids established from the control mice, suggesting that this colonoid culture methodology does not appropriately reflect the in vivo metabolic changes that are observed in mice15.
The failure of PGC1α to initiate mitochondrial biogenesis during an intestinal inflammatory insult is primarily localized to the differentiated colonic epithelium15,19. To mirror the changes observed in vivo, we decided to direct the colonoids toward a more differentiated state. To induce this shift, the colonoids were initially incubated with traditional colonoid medium. After 24 h to 48 h, the colonoids were switched to differentiation medium for an additional 48 h. During differentiation, a small percentage of colonoids transitioned from a spherical architecture into budded structures enriched with goblet cells and colonocytes17 (Figure 2A,B). The colonoids grown in WRN-supplemented medium were enriched with rapidly dividing Lgr5+ stem cells. To confirm that both the Lgr5+ stem cells and other proliferating cells exited the cell cycle during differentiation, the mRNA levels were analyzed via qPCR. Significant downregulation in both Lgr5 (97%, p = 0.0023) and Ki67 (75%, p = 0.0007) levels was observed in the differentiated colonoids (n = 6) compared to the colonoids grown in a traditional culture setting (n = 6; Supplementary Figure 2A,B). One would also expect the differentiated colonoids to be primarily comprised of mucus-secreting goblet cells. Additionally, in fact, MUC2 was upregulated nearly two-fold in the differentiated colonoids compared to their traditionally grown counterparts (n = 3, p = 0.0433, Supplementary Figure 3). When taken together, these data suggest that the colonoids were successfully differentiated and were ready for downstream experimentation. Next, inflammatory mediators were added into the differentiation medium, as described in section 5 of the protocol. Protein and RNA were collected up to 72 h after the introduction of the inflammatory mediators for analysis. However, at 72 h, increased cell death was occasionally observed.
To assess whether the introduction of inflammatory mediators to differentiated colonoids could mimic the mitochondrial biogenesis failure observed in both human IBD and an acute model of murine colitis, the protein expression of PGC1α and transcription factor A, mitochondria (TFAM) was assessed via western blot analysis in differentiated colonoids (n = 5) treated with inflammatory mediators over 72 h. PGC1α expression has been shown to be decreased in murine models of colitis, as well as in human IBD15. TFAM, a protein that drives both the transcription and replication of the mitochondrial genome, has also been shown to be decreased in murine models of colitis, and its gene expression has been shown to be decreased in human IBD15,20. We observed a similar downregulation in both PGC1α (50.4%, p = 0.0442) and TFAM (88.9%, p = 0.004) expression in the differentiated colonoids after 72 h of exposure to these inflammatory mediators (Figure 3). Collectively, this suggests that the treatment of differentiated colonoids with cytokines is an ideal model to study mitochondrial dynamics in vitro.
While various cellular and molecular functions can be examined in 3D colonoid culture settings, 2D monolayer culture systems are superior when assessing barrier function, host-pathogen interactions, and the impact of orally absorbed therapeutics on inflammation21,22. The formation of a continuous layer of confluent cells with an intact barrier allows different biological or chemical agents to be placed on the apical and/or basal side of the cells with ease. This protocol allows for 2D monolayer cultures to be established from pre-existing WT colonoids that have been passaged twice. Colonoids that have been enzymatically and mechanically disrupted are plated on cell culture inserts coated with basement membrane matrix. Upon initial plating, the fragmented colonoids appear in cellular clusters ranging from 15 to 150 cells (Figure 4A). By Day 3, the cells have begun to flatten out and slowly cover the cell culture insert, generally reaching confluency (Figure 4B). Over the next couple of days, monolayers continue to grow and form a continuous barrier that can be quantitatively measured by TEER (Figure 4C,D). Interestingly, the TEER measurements fluctuate over Day 3 to Day 5. The monolayers have higher values earlier on with a broad range (200-800 Ω·cm2), but these values slowly converge on a lower number by Day 5 (115-150 Ω·cm2, Figure 5A). When the monolayers reach a steady state, they can be used for downstream experimentation.
To characterize the epithelial response of colonoid-derived monolayers to inflammation, inflammatory mediators were placed on the basal side (the outside of the insert) of the cells for 48 h. The RNA was extracted, and the mRNA levels of various stem and cell differentiation markers, as well as junctional proteins, were analyzed via qPCR. Lgr5 was not detected in either condition, but another stem cell marker, mTERT, was reduced by nearly 60% in the inflamed monolayers compared to the untreated control (Supplementary Figure 4A). Next, we analyzed the expression of two markers of differentiated cell types, Muc2 and Alkaline phosphatase (Alpi). Both mRNA transcripts were lower in the inflamed monolayers compared to the control cells (Supplementary Figure 4A). Both stem and post-mitotic cells are commonly decreased during colonic inflammation23,24,25, and a similar response was observed in the monolayer model obtained in this study.
A benefit of the monolayer system is the ability to assess the barrier response. To determine whether the barrier was compromised in the immune-stimulated condition, we measured the TEER values after 48 h. The TEER values for the control monolayers (n = 2) averaged 129.16 Ω·cm2 but dropped significantly (p = 0.0087) to an average of 98.54 Ω·cm2 in the inflamed monolayers (Figure 5B). To further confirm that the barrier was compromised in the inflammatory condition, we assessed the mRNA levels for three different tight junction markers, Zo-1, Occludin, and Claudin. All three markers had reduced levels in the inflamed condition compared to their uninflamed counterparts (Supplementary Figure 4B). Collectively, these data show that barrier dysfunction is present in the inflamed monolayers and mimics the barrier dysfunction observed during IBD pathogenesis.
Figure 1: Initial isolation of murine crypts for the establishment of long-term colonoid culture. (A) On Day 0, 300-500 crypts were plated in basement membrane matrix (picture obtained 5 h after plating), and the growth of the colonoids was captured on (B) Day 1, (C) Day 3, and (D) Day 5. On Day 5, the colonoids were ready to be passaged. Magnification = 10x; scale bar = 400 µm. Please click here to view a larger version of this figure.
Figure 2: Establishment of terminally differentiated colonoids from traditional colonoid culture. Differentiation medium was placed on the colonoids 48 h after passaging the murine colonoids. After (A) 24 h and (B) 48 h, the colonoids become enriched with terminally differentiated cells. Magnification = 10x; scale bar = 400 µm. Please click here to view a larger version of this figure.
Figure 3: PGC1α and TFAM protein expression in murine differentiated colonoids treated with inflammatory mediators. Medium supplemented with inflammatory mediators was placed on the differentiated colonoids, and protein was collected 0 h, 24 h, 48 h, and 72 h after exposure. (A) The protein expression of PGC1α showed an overall significant decrease (p = 0.0442) over 72 h. TFAM was significantly downregulated (p = 0.004) after exposure to inflammatory mediators at 72 h, and (B) there was a downward trend in protein expression at 24 h, 48 h, and 72 h when analyzed by ANOVA. Data presented as mean ± standard deviation. *p < 0.05, **p < 0.005. Please click here to view a larger version of this figure.
Figure 4: Establishment and growth of monolayers from pre-existing colonoids. Colonoids that had been enzymatically and mechanically disrupted were plated on cell culture inserts coated with a basement membrane matrix. Upon initial plating, the fragmented colonoids appeared in (A) cellular clusters, and by (B) Day 3, they had begun to flatten out and slowly cover the cell culture insert. (C) At Day 5 and (D) Day 7, the monolayers continued to grow to form a continuous barrier that could be quantitatively measured by TEER. Magnification = 10x; scale bar = 400 µm. Please click here to view a larger version of this figure.
Figure 5: TEER values for establishing colonoid-derived monolayers and inflamed monolayers. (A) The TEER values were measured using a voltohmmeter from Day 3 to Day 5. The monolayers had higher values earlier on with a broad range but slowly converged on a lower number by Day 5. Once the monolayers reach a steady state, they can be used for downstream experimentation. (B) The TEER values were lower in the inflamed monolayers (n = 2) compared to the uninflamed controls (n = 2). The control monolayers had an average TEER of 129.16 ohms·cm2, and the TEER of the inflamed monolayers was significantly lower (p = 0.0087), with an average of 98.54 Ω·cm2, when analyzed using a t-test. **p < 0.01. Please click here to view a larger version of this figure.
Table 1: Solution composition table. Please click here to download this Table.
Supplementary Figure 1: PGC1α protein expression in murine colonoids grown in traditional colonoid medium isolated from WT controls and 2% DSS-treated mice. The colonoids were derived from both 8-week-old WT control mice and 2% DSS-treated mice after 7 days. Protein was isolated from colonoids passaged twice and on Day 4 after plating. There was no difference in PGC1α protein expression (p = 0.9996) in the colonoids derived from the control mice compared to the mice exposed to 2% DSS for 7 days when analyzed by an unpaired t-test. Data presented as mean ± standard deviation. Please click here to download this File.
Supplementary Figure 2: Reduced Lgr5 and Ki67 mRNA expression in differentiated colonoids. Murine colonoids passaged 48 h prior were exposed to differentiation medium for 48 h before isolating the RNA and cDNA synthesis. The mRNA levels were determined via qPCR and analyzed by the comparative CT method. (A) Lgr5 and (B) Ki67 were significantly decreased in the differentiated colonoids when statistically analyzed using a t-test (p = 0.0023, p = 0.0007). Data presented as mean ± standard deviation.**p < 0.005. Please click here to download this File.
Supplementary Figure 3: Increased mucin-secreting goblet cells in differentiated murine colonoids. Murine colonoids passaged 48 h prior were exposed to differentiation medium for 48 h. The cells were lysed in RIPA buffer, and protein was visualized by western blot analysis. (A) MUC2 expression was increased in each set of differentiated colonoids compared to the undifferentiated colonoids. (B) A nearly two-fold change in MUC2 protein was observed in the differentiating conditions, which was significant when analyzed using a t-test (p = 0.0433). Data presented as mean ± standard deviation.*p < 0.05. Please click here to download this File.
Supplementary Figure 4: Reduced expression of stem and differentiation markers, as well as tight junctional markers, in monolayers treated with inflammatory mediators. Murine colonoid-derived monolayers (Day 5) were exposed to inflammatory mediators for 48 h, RNA was subsequently collected, and cDNA was synthesized (n = 2). The mRNA levels of stem, differentiation, and junctional markers were determined via qPCR and analyzed by the comparative CT method. (A) The stem cell marker, Lgr5, was not detected in either condition, but mTERT was substantially reduced in inflammatory conditions. The goblet cell marker, Muc2, and the colonocyte marker, Alpi, were both decreased in the inflamed monolayers compared to the untreated monolayers. (B) The tight junction markers, Zo-1, Occludin, and Claudin, were all lower in inflammatory-treated monolayers compared to the control. Data presented as mean ± standard deviation. Please click here to download this File.
Supplementary Table 1: List of primers. Please click here to download this File.
Organoid development has revolutionized the way the scientific community studies organ systems in vitro with the ability to partially recapitulate cellular structure and function from an animal or human in a dish. Further, organoid systems derived from humans with diseases offer a promising tool for personalized medicine that could guide therapeutic decision-making. Here, we describes a crypt isolation protocol that works well and introduces key steps that allow for cleaning up excess debris in the isolation before plating. Additionally, we explain a key detail that is often overlooked in the process–the ability to accurately count the crypts for plating. Once the colonoids are established, we outline key manipulations that allow for their cellular differentiation or monolayer formation, which could enhance the ability to study changes in the intestinal epithelium during IBD.
The crypt-villus axis in the colonic epithelium in mammals is a complex structure composed of different cell types with different functions. The maintenance of this axis hinges upon both intrinsic and extrinsic cellular factors, which together help maintain epithelial homeostasis18. We have noted that murine colonoids grown in traditional medium primarily reflect stem cells as opposed to the conglomeration of both stem cells and terminally differentiated cells that is seen in vivo. Moreover, murine colonoids grown and maintained in this manner are circular structures as opposed to flat monolayers. These differences have potential implications for our attempts to study intestinal disease in vitro. Specifically, we are limited in our ability to use organoids to study changes in barrier function, as well as the effects of inflammation on terminally differentiated intestinal epithelial cells.
Here, we describe protocols for not only the isolation of murine crypts to grow colonoids but also for the manipulation of these colonoids to study intestinal epithelial physiology and the epithelial response to inflammation. Among several extrinsic factors that promote the gradient of pluripotent stem cells and terminally differentiated cells is the Wnt canonical pathway, which is rich in crypts and promotes stem cell health. Wnt signaling diminishes at the tip of the crypt axis, coinciding with the evolution of stem cells into terminally differentiated cells26. We have described a protocol that allows for the terminal differentiation of colonoid cells. Like others, we differentiate by removing Wnt from the growth medium, but we use a lower concentration of R-spondin (500 ng/mL versus 1 µg/mL R-spondin)17. Surprisingly, a 50% reduction in R-spondin does not impact the successful differentiation of the organoids. The ability to differentiate with a lower concentration helps in establishing a less artificial system that might more closely resemble the physiological environment in vivo. Although this protocol results in finite terminal differentiation (Supplementary Figure 2 and Supplementary Figure 3) and an inability to maintain cellular immortality, it is nonetheless a useful method for better understanding the physiologic contribution of the terminally differentiated epithelium, including colonocytes, enteroendocrine cells, and goblet cells, to IBD and other disease states. Furthermore, we show that supplementing specific cytokines into this medium, which are traditionally elevated in patients with IBD, directs the differentiated organoid system toward an inflammatory state. Over the course of 72 h, differentiated colonoids treated with inflammatory cytokines more closely reflect some of the metabolic findings shown in other established models of colitis and human IBD (Figure 3). Furthermore, differentiated colonoids treated in this manner are likely more reflective of the effects of inflammation on cellular homeostasis on the differentiated epithelium. We have also outlined a protocol for developing monolayers, which enables the formation of both an apical and basal side of the epithelium, allowing for an analysis of barrier function in vivo. This system is useful in studying mechanisms of barrier dysfunction without the need for differentiation and cultivating new therapeutic targets for treatment.
Several limitations and potential pitfalls regarding the protocols described above must be noted. The successful isolation of crypts and the development of colonoids can be limited by the crypt yield from each animal, the accuracy of counting the crypts, and the cleanliness of the prep. The speed of counting the isolated crypts after resuspension, variations in the mechanical dissociation of the crypts from the underlying muscularis, and the number of washes and centrifugal spins contribute to successful colonoid culture. Furthermore, it is critical not to fully disassociate the organoids into single cells during the passaging or plating for 2D monolayer culture. Clusters of 30 to 50 cells are ideal to allow the culture to propagate for long-term culture. The above protocols note areas of potential pitfalls and optional additional steps to troubleshoot these issues. Ultimately, the protocol may require optimization by the individual performing the isolation due to inherent variability in skill set and maneuvers (i.e., shaking), as well as variation in each animal. Additional optimization might be required by the end user to capture the genetic and molecular diversity found in IBD.
Ultimately, while the colonoid system is a highly useful tool for recapitulating key aspects of human physiology and disease in vivo, this system is ultimately limited in its ability to study all aspects of human disease. Traditional methods for colonoid development have elucidated key aspects of stem cell dynamics; however, these findings often do not adequately reflect the findings in terminally differentiated cells within the host epithelium. The strategies outlined in this study provide investigators with a relatively fast, inexpensive model for organoid manipulation that, when employed properly, can shed light on key aspects of intestinal epithelial structure and function during inflammation.
The authors have nothing to disclose.
This work was supported by National Institutes of Health Grants R01DK120986 (to K.P.M.).
0.4-μM transparent transwell, 24-well | Greiner Bio-one | 662-641 | |
15-mL conical tubes | Thermo Fisher | 12-565-269 | |
50-mL conical tubes | Thermo Fisher | 12-565-271 | |
70-μM cell strainer | VWR | 76327-100 | |
Advanced DMEM/F12 | Invitrogen | 12634-010 | Stock Concentration (1x); Final Concentration (1x) |
B-27 supplement | Invitrogen | 12587-010 | Stock Concentration (50x); Final Concentration (1x) |
Chopsticks Electrode Set for EVO | World Precision Instruments | STX2 | |
Corning Matrigel GFR Membrane Mix | Corning | 354-230 | Stock Concentration (100%); Final Concentration (100%) |
Dithiothreitol (DTT) | Sigma-Aldrich | D0632-5G | Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water) |
DMEM high glucose | Thermo Fisher | 11960-069 | Stock Concentration (1x); Final Concentration (1x) |
Dulbecco's phosphate-buffered saline without Calcium and Magnesium | Gibco | 14190-144 | Stock Concentration (1x); Final Concentration (1x) |
Ethylenediaminetetraacetic acid (ETDA) | Sigma-Aldrich | E7889 | Stock Concentration (0.5 M); Final Concentration (30 mM) |
Fetal Bovine Serum | Bio-Techne | S11150H | Stock Concentration (100%); Final Concentration (1%) |
Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm | Thermo Fisher | 12-550-15 | |
G418 | InvivoGen | ant-ga-1 | Final Concentration (400 µg/µL) |
Gentamicin Reagent | Gibco/Fisher | 15750-060 | Stock Concentration (50 mg/mL); Final Concentration (250 μg/mL) |
GlutaMAX-1 | Fisher Scientific | 35050-061 | Stock Concentration (100x); Final Concentration (1x) |
HEPES 1 M | Gibco | 15630-080 | Stock Concentration (1 M); Final Concentration (10 mM) |
hIFNγ | R&D Systems | 285-IF | Stock Concentration (1000 ng/µL); Final Concentration (10 ng/mL); Solvent (ultrapure water) |
hIL-1β | R&D Systems | 201-LB | Stock Concentration (10 ng/µL); Final Concentration (20 ng/mL); Solvent (ultrapure water) |
hTNFα | R&D Systems | 210-TA | Stock Concentration (10 ng/µL); Final Concentration (40 ng/mL); Solvent (ultrapure water) |
Hydrogen Peroxide | Sigma | H1009 | Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media) |
Hygromycin B Gold | InvivoGen | ant-hg-1 | Final Concentration (400 µg/µL) |
L-WRN Cell Line | ATCC | CRL-3276 | |
mEGF | Novus | NBP2-35176 | Stock Concentration (0.5 µg/µL); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA) |
N-2 supplement | Invitrogen | 17502-048 | Stock Concentration (100x); Final Concentration (1x) |
N-Acetyl-L-cysteine | Sigma | A9165-5G | Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water) |
Noggin | Peprotech | 250-38 | Stock Concentration (0.1 ng/µL); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA) |
Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher | 15140-122 | Stock Concentration (100x); Final Concentration (1x) |
Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable | VWR | 25384-342 | |
Polystyrene Microplates, 24 well tissue culture treated, sterile | Greiner Bio-one | 5666-2160 | |
R-Spondin | R&D Systems | 3474-RS-050 | Stock Concentration (0.25 µg/µL); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA) |
Tryp LE Express | Thermo Fisher | 12604-013 | Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA) |
UltraPure Water | Invitrogen | 10977-023 | Stock Concentration (1x); Final Concentration (1x) |
Y-27632 dihyddrochloride | Abcam | ab120129 | Stock Concentration (10 mM); Final Concentration (10 µM); Solvent (UltraPure Water) |