Immunology and Infection
Published: January 27th, 2019
This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.
Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.
Sepsis is a leading cause of human newborn infectious deaths1. Because newborn sepsis is poorly understood, little progress has been made in both the identification of at-risk newborns early during the disease and the development of efficacious treatments or prophylaxes. This necessitates the use of animal models of sepsis to better understand the process and test possible interventions. Furthermore, adult rodents respond differently to sepsis, with statistically significant differences in the number of bacteria to administer to obtain the same lethal dose (LD) and differences in the resulting host response as compared to newborns2. Thus, neonatal sepsis has to be studied in neonates. Several adult sepsis models have been used in sepsis research. These include an intravenous challenge with specific organisms implicated in adult human sepsis or cecal ligation and puncture (CLP). CLP is an endogenous challenge model where the cecum is surgically isolated, ligated, and punctured to allow leakage of intestinal contents into the peritoneum, eventually leading to the systemic dissemination of microbes and their products3. However, the surgical procedure required to establish CLP is lethal to newborn animals; therefore, an alternate method is necessary to mimic the polymicrobial challenge of CLP to induce neonatal sepsis. The cecal slurry model for neonatal polymicrobial sepsis was developed to address this need, whereby the cecal contents of animals are harvested, suspended in sterile dextrose 5% in water (D5W), and intraperitoneally injected into newborn mice2. This has, since, become an increasingly popular model to study sepsis in both newborn and adult animals and has substantially advanced mechanistic insights in the disease's process4,5,6,7,8,9,10,11,12,13,14,15.
Given the increasing use of this model and desire of researchers to directly compare results across publications, there is a need for the technical aspects to be well described and standardized across studies. Standardization applies to three aspects of the model, namely, i) the preparation of the cecal slurry stock, ii) the preparation of the challenge aliquots for injection into the experimental animals, and iii) the definition of the humane endpoint whereby animals are deemed nonsurvivors in challenge experiments. Specifically, methods to prepare the cecal slurry stock are often referenced to the original article introducing the model2. A brief summary of that model is that cecal contents from adult mice were harvested, suspended in sterile D5W to a concentration of 80 mg/mL, and used within 2 h to inject the experimental animals. This original model used mice of the same age, from the same vendor location, which were housed in their respective research facilities for less than 2 weeks prior to harvesting cecal contents. The use of in-house bred mice, although reducing the cost from regular vendor delivery and allowing for the use of excess mice of a broader range of sex and age, also substantially increased donor-to-donor variability. This motivated the development of an alternative technique, whereby cecal contents from multiple mice were pooled together to prepare a large stock, which was then aliquoted and stored at -80 °C13. This alternate method was adapted by multiple groups14,15. However, that adaptation resulted in some technical variations, both in the storage media used (10% or 15% glycerol, or D5W alone) and in the strategy of filtration to remove particulate (multistage filtration through a 860 µm and, then, a 190 µm filter, or individual filtrations through 100 µm or 70 µm filters)13,14,15. The injection of glycerol alone could potentially cause harm, given that 25%-50% glycerol injections have been used as a rodent model of renal injury16,17,18,19,20. To avoid unintended side effects of glycerol, the cecal slurry stock preparation for mice in this study is frozen in D5W without glycerol, and tests of bacterial viability from storage at -80 °C are performed. The filtration strategy used in this study is one pass through a 70 µm filter, which has not been directly compared to the other filtration strategies listed.
Lethal weight-adjusted doses of injected cecal slurry may vary from facility to facility and should be titered out to the desired lethality for individual groups. With different challenge doses, the accompanying challenge volumes change by necessity. However, this methodological detail has not been reported before. Furthermore, strategies for standard procedures, such as intraperitoneal injection, are rarely elaborated on within the literature, but individual techniques may affect whether newborn mice leak when injected and impact their final outcomes.
Animal welfare, including a definition of humane endpoint, is a central aspect of this model and in any model of infection and inflammation in rodents21. In 1998, the Canadian Council on Animal Care (CCAC) published extensive guidelines for humane endpoint selection, defining the humane endpoint as "any actual or potential pain, distress, or discomfort should be minimized or alleviated by choosing the earliest endpoint that is compatible with the scientific objectives of the research"22. Others also caution that humane endpoints must be established based on scientific justification rather than on a subjective interpretation of the animal's state alone21. While there is a wealth of resources for clinical, behavioral, and body-condition sign-based criteria for humane endpoint, even in the context of infection and inflammation specifically21,23,24, none of these, including the CCAC guidelines for humane endpoint22, mention newborn mice. Thus, objectively and scientifically justified humane endpoints are much more difficult to establish for newborn animals, given both their limited behavioral capabilities and the lack of evidence from criteria like weight loss, which is commonly used for adult mice. Currently, the criteria for the humane endpoint used for 5- to 12-day-old neonatal mice in the cecal slurry literature all reference back to the original manuscript that introduced the model2. In this original paper, the definition of humane endpoint for newborn animals was based on two criteria; namely, the location of a mouse outside of the nest (scattering) and the lack of milk spots had been seen to result in death within hours. A complicating matter in assigning a humane endpoint is that milk spots become difficult to see in mouse strains with dark fur, such as the commonly employed C57BL/6J strain, after the first week of life, while sick animals are monitored until the 14th day of life (DOL). Further, dead animals can be found postchallenge when applying these criteria (own observation; unpublished); thus, a more rigorous definition of humane endpoint is necessary to alleviate suffering to experimental animals and avoid mortality in situations where the outcome could be accurately discerned earlier.
All three methodological aspects of the cecal slurry model are presented in a standard operating procedure detailing the preparation of cecal slurry stock, a method for injecting experimental animals that keeps the injection volume constant between doses and reduces the risk of leaks, and a definition of humane endpoint for 7- to 12-day-old mice based on a system of behavioral modeling. Behavioral information of mouse health scores from over 240 experimental animals was collected and grouped by final survival outcome, demonstrating an evidence-driven definition of humane endpoint. The suffering of experimental animals is reduced by identifying moribund neonatal mice at the earliest possible time point, while biologically significant survival outcomes can be inferred by observing key variables. The visual representation of both cecal slurry preparation and neonatal mouse behaviors will serve as an excellent resource to any group studying sepsis or newborn challenge model animals.
All experiments in this protocol have been approved by the University of British Columbia Animal Care Committee under protocol number A17-0110.
1. Tool sterilization
2. Cecal slurry preparation
3. Sepsis challenge of 7-day-old neonatal mice
4. Mouse monitoring
5. Titration of the cecal slurry
Cecal slurry viability stored at -80 °C can be tested over time by serially diluting and plating aliquots of cecal slurry stock on 5% sheep's blood tryptic soy agar followed by 24 h of aerobic incubation at 37 °C. Subsequent counting of culturable colony-forming unit (CFU) content of a cecal slurry preparation was found not to change over a 6 month period, and the viability was not affected by prolonged storage at -80 °C (Figure 2). Each donor mouse resulted, on average, in enough cecal slurry to challenge three to four litters (data not shown).
Mice challenged at DOL 7 with cecal slurry to induce polymicrobial sepsis began to reach the humane endpoint within 12 h of the challenge, and polymicrobial sepsis was mostly resolved by 48 h postchallenge, as observed in a Kaplan-Meier survival curve combined from data from over 200 challenged mice (Figure 1A). The lethality was dependent on the challenge dose administered, with a 5% change in challenge dose resulting in a roughly 15% difference in survival rate (Figure 1B). The mouse body weight was measured at each monitoring visit. Weight loss was seen in all challenged animals, being nondiscriminatory between mice that ended up surviving and those that did not during the initial 24 h postchallenge (Figure 1C). After 24 h, most surviving animals began to regain their weight, while all nonsurvivors continued to lose weight and moved to their humane endpoint. However, a small proportion of surviving animals that had retained their righting reflex also continued to lose weight or failed to gain weight, until the end of the experiment, even losing as much as 20% of their initial body weight within 40 h of the challenge. As there was an overlap of weight loss between mice that ended up surviving and those that did not, the change in weight or a threshold of weight loss could not be used as a criterion for humane endpoint while still maintaining the goal of accurately dividing survivors from nonsurvivors.
The behavior of mice was monitored as outlined in the protocol and in Table 2. Snapshots of the health categories are displayed (Figure 3A-C). These photos show the different health categories of mice who failed to right themselves after being placed on their back and outline the difference between FTR-Mobile and FTR-Lethargic, which is an important distinction. Unchallenged healthy mice of this age do not display FTR-Lethargic activity; therefore, this health category is a marker of disease and a response to challenge. Sick mice displayed FTR-Lethargic symptoms (Figure 3B) and could regress toward FTR-Nonmobile (Figure 3C), where the upper leg remains parallel with the bottom leg, with little to zero hip rocking movement, which is one of the criteria for humane endpoint. The mice might also recover, gaining increased hip movement and becoming FTR-Mobile (Figure 3A). The righting reflex and mobility scores were determined for both the left and right side of each mouse, and the highest score was utilized to determine whether the mouse had reached a humane endpoint. Behavioral information was collected from over 240 animals challenged with a lethal dose 60 (LD60) of cecal slurry, and 144 humane endpoints were observed (Figure 3D-F and Table 1). This evidence-driven approach was used to define and refine the humane endpoint across four disease stages, categorized by the experimenters based on both behavioral differences between survivors and nonsurvivors and by the fraction of humane endpoints reached during each time frame. During early experiments, FTR-Nonmobile mice that had no hip movement were consistently found dead within 4-6 h of this behavior being observed. In the collection of the presented information, an FTR-Nonmobile health score was used as criterion for a humane endpoint. From 12-21 h postchallenge, while FTR-Nonmobile mice were euthanized, both surviving and nonsurviving animals displayed very similar behavioral patterns and could not be distinguished in any other way (Figure 3D). From 21-48 h postchallenge, the majority of surviving mice regained their righting reflex, while fewer than 1% of the FTR behaviors observed were in animals that would go on to survive the experiment (Figure 3E). Thus, mice that failed to right themselves from both sides became an additional criterion for humane endpoint during this time. Between 12 and 20 h postchallenge, 12.5% of the total number of humane endpoints were observed, versus 80.5% between 20 and 48 h, and 7% after 48 h (Table 1). A distinguishing feature between mice that ended up surviving and that eventually worsened to a humane endpoint was the loss of the righting reflex, independent of hip mobility (Figure 3F). Indeed, between 20 and 48 h after the challenge, a total of 121 mice had failed to right themselves from both sides, with 116 of these mice eventually progressing to a humane endpoint (which represents a 96% accuracy in identifying mice that would not recover). Beyond 48 h after the challenge, 11 mice were observed to fail to right themselves from both sides, and 10 of these progressed to a humane endpoint (a 91% accuracy). Beyond 20 h after the challenge, the number of mice that lost the righting reflex for both sides predicts the final outcome with an accuracy of more than 90%; therefore, this has been added to the humane endpoint criteria, to identify nonrecovering mice earlier and reduce mouse suffering (Table 1).
The frequency that mice need monitoring changes over time, due to different rates of death postchallenge, and is outlined in Table 1. A mouse was considered to be at its humane endpoint at any point if it had failed to right itself and displayed nonmobile hip movement on both sides, or if the mouse was found scattered from the nest, was unable to right itself, and had lethargic hip movement. Mice with either of these conditions were not expected to be able to rejoin the litter and have been observed to be FTR-Nonmobile within 4-6 h. Starting 20 h after the challenge, a new humane endpoint was added because the presented information shows that the vast majority of mice that FTR from both sides ends up succumbing to disease.
The videos, tables, and resources presented in this manuscript are an effective teaching resource for the correct behavioral assignment of challenged mice. Seven experimenters were asked to watch the training video and read both the protocol and the tables before assigning behaviors to 60 challenged animals. The identification of humane endpoint assignment was accurate both for distinguishing FTR-Nonmobile mice from mice that displayed the other behaviors (Figure 4A) and FTR mice from mice that were able to right themselves within the allowable time frame (Figure 4B).
Figure 1: Kaplan-Meier survival curve, cecal slurry dose titration, and weight change following the cecal slurry challenge. (A) Survival outcome of neonatal C57BL/6J mice challenged with an intraperitoneal cecal slurry injection at DOL 7. The data for this figure were combined from independent experiments using multiple challenge doses, ranging from 0.7 to 1.3 mg of cecal slurry per gram body weight was administered to these mice. (B) Neonatal mice challenged with 0.80 to 0.95 mg of cecal slurry per gram body weight from one cecal slurry preparation display a dose-dependent relationship between the amount of cecal slurry given and the percentage of survival.(C) The percentage of change in weight compared to the challenge weight, with the dotted line denoting a 20% loss of weight from the time of the challenge. Please click here to view a larger version of this figure.
Figure 2: CFU concentration in cecal slurry stock stored at -80 °C does not change over a 6 month period. The effect of the cecal slurry age on CFU concentration was tested using linear regression. Each point represents one aliquot of the same cecal slurry preparation, serially diluted and plated over a 6 month period. Please click here to view a larger version of this figure.
Figure 3: Hip mobility categories of mice that fail to right themselves and of animal behaviors at various times postchallenge. Mice that have been challenged with sepsis, when placed on their back, will display signs of morbidity that can be measured by the degree of hip movement. (A) A fail to right (FTR)-Mobile mouse shows hip rocking movement of their upper leg exceeding 90° angle from horizontal. (B) An FTR-Lethargic mouse shows hip rocking movement but does not exceed 90° angle from horizontal at any point during the 4 s of monitoring. (C) Some FTR-Nonmobile mice will extend their leg, bending at the knee, but will show very little (less than 10° angles) to zero hip rocking movement, and the legs will remain parallel to each other. (D) Animal behaviors 12-21 h postchallenge show that only FTR-Nonmobile behaviors separate survivors from nonsurvivors. (E) From 21 to 48 h postchallenge, only 4 out of the 592 observed FTR behaviors (0.67%) belong to survivors, allowing the righting reflex to predict the final outcome and be used as a new criterium for humane endpoint. (F) Beyond 48 h postinfection, 6 out of 131 mice (4.55%) that had a righting reflex went on to become part of the FTR group and were sacrificed by the end of the experiment, justifying sustained monitoring throughout the course of recovery. Please click here to view a larger version of this figure.
Figure 4: Instructional resources result in accurate behavioral classification by independent experimenters. Experimenters trained by watching video accompanying this protocol categorized videos of 60 neonatal mice into different health groups. (A) The ability to distinguish a humane endpoint was determined and an average of 97% of behaviors was accurately categorized as FTR-Nonmobile or not, while only 1% of FTR-Nonmobile mice were misidentified. Two percent of the mice were falsely identified as FTR-Nonmobile. (B) The identification of the second humane endpoint criterium of correctly distinguishing between FTR mice or those having the ability to right themselves within 4 s of being placed on their back was assigned correctly in 97% of the scorings, while only 0.96% of the mice were incorrectly assigned as righting themselves and 2% of mice were incorrectly assigned as FTR. Please click here to view a larger version of this figure.
|A: High morbidity, no mortality
|B: High morbidity, low mortality
|C: High morbidity, high mortality
|D: Low morbidity, low mortality
|Hours post challenge
|2 h post challenge
|every 4−6 h
|Every 4−6 h, 8 h, unattended overnight
|1−2 times daily, more if needed
|Proportion of total humane endpoints observed
|Percentage of humane endpoints observed
|Humane endpoint criteria
|1. FTR−Nonmobile on both sides
|1. FTR−Nonmobile on both sides
|2. Scattered from nest and is FTR−Lethargic
|2. Scattered from nest and is FTR−Lethargic
|3. FTR on both left or right side (with any mobility score)
Table 1: Frequency of monitoring and humane endpoint criteria in the different stages of disease. Monitoring frequency, humane endpoints observed, the percentage of humane endpoints, and humane endpoint criteria during different stages of disease.
|Time limit to right after being placed on back
|Time limit to measure amount of movement (mobile / lethargic / nonmobile)
|Mobility scoring Criteria
|An additional 8 s
|The mouse takes multiple steps in a row, maintaining forward momentum, and explores its environment. Pup will not fall over.
|The mouse can take a step but will stop and pause before taking another. Pup may fall over.
|The mouse does not take any steps after righting itself. Pup may fall over.
|Fail to right
|The same 4 s used to measure righting reflex
|Has energetic hip movement with the upper leg rotating beyond 90° from horizontal at least once within 4 s.
|Hip movement up to but not beyond 90° from horizontal.
|Limbs may move by extending and retracting but the hips will not rotate. Pup looks very sickly.
Table 2: Monitoring table and criteria in determining the health score of mice. The provided criteria were used to define health category groups to mice, and to reduce individual variance in assigning health scores.
Postnatal neonatal mice have very limited mobility and fail to right themselves after being placed on their back, even when unchallenged. By DOL 7, the age of mice challenged in this model, a range of movement spanning from Rights-Mobile to FTR-Mobile was observed in unchallenged mice, with an important difference, namely that an unchallenged mouse at this age did not display FTR-Lethargic behavior. Only mice challenged with polymicrobial sepsis were observed to become FTR-Lethargic; therefore, this response can be a marker of disease severity. Being attentive to the cutoff of a 90° angle from horizontal for hip movement allows for the consistent and accurate assignment of lethargic or mobile hip movement in mice. The time frame of 4 s to see if a mouse can right itself was selected because unchallenged mice were able to consistently right themselves within this time frame. Repeated measurement of the same mouse was avoided, while the time to right themselves and the measurement of hip mobility was limited to 4 s, to avoid excessively tiring the mouse, which could otherwise affect its ability to obtain food and warmth and could affect its prognosis to get better. Righting itself from both the left and the right side were observed, and the higher of the scores was used to determine if the mouse was at a humane endpoint, because some mice were found to display FTR-Nonmobile on one side yet have a higher mobility on the other side and be able to recover eventually.
The scoring system used to evaluate mouse health relied on the application of categorical cutoffs to what is a spectrum of movement and, therefore, could be prone to individual bias. Staff was trained together to ensure each person scored the mice the same; however, there will likely remain a level of subjectivity leading to variation. The consistency of scoring was evaluated by having seven researchers who had not previously performed the neonatal mouse monitoring learn the requirements outlined in this protocol and video and, then, independently assign behaviors and determine humane endpoint. A 97% accuracy was observed with scoring performed on 60 challenged mice, suggesting that individual bias does not play a substantial role in the behavioral assignments of this model. The presented behavioral monitoring protocol is based on observations of animals challenged on DOL 7, yet mice younger than 6 days in an unchallenged healthy state cannot consistently right themselves. Thus, the described humane endpoint criteria could not be applied directly to younger mice. If younger mice are used in this experimental model or if a different challenge model with different disease kinetics is applied, then suitable humane endpoint criteria must be developed and piloted to avoid the euthanasia of mice that would otherwise, eventually, recover. The scoring system displays a robust method of improving humane endpoint classification that, with testing and confirmation, could potentially be applied to other models.
Each preparation of cecal slurry or the use of a new mouse strain required the retitration of the cecal slurry dose to administer to achieve a similar lethal dose. Each preparation was standardized by the readout of interest, namely survival, rather than giving the same bacterial count. Each cecal slurry preparation's viable bacterial concentration varied slightly, potentially due to differences in the donor's commensal bacteria or due to variances in the weight left in the cell strainer of the cecal slurry stock postfiltration. During the titration of the cecal slurry, the first two litters were divided into two groups and each half of the litter were challenged with one of the two doses so that each of the doses would be tested in two litters. If the resulting survival rate did not match the required level, then the challenge dose was either increased or decreased by 5%-10% and the experiment repeated. Multiple litters were used to account for litter-to-litter differences that could cause resistance or increased susceptibility to sepsis across a litter. It was important to accurately titer the cecal slurry stock with each new preparation to ensure that the new titration of cecal slurry was comparable to previous cecal slurry preparations. Periods of excess noise and vibration, specifically during the compacting of asphalt and the construction of a nearby building and road, were observed to increase stress in the dams. This correlated with increased rates of cannibalization, and affected the mortality of the survival experiments, even affecting unchallenged mice, indicating that there can be extraneous impacts on neonatal survival that also need to be controlled for.
Prior methods for cecal slurry stock preparation included either the use of fresh cecal slurry or the preparation of frozen cecal slurry, using a variety of methods, including the storage in glycerol that would inevitably be transferred during the challenge. While the use of fresh cecal slurry provides the advantage of having a bacterial composition closest to original cecal contents, there is the risk of variance between individual donor mice due to the variation of commensal bacteria. While this was minimized by using cecal donors from the same vendor with minimal time between arrival and progression of the experiment, this could become a cost-prohibitive option for some laboratories and presented another timing logistics challenge in having age-matched mice available when commencing a cecal slurry experiment in neonatal mice that were 7 days old. An alternative method to using fresh cecal slurry was utilized, where multiple adult donors' cecal contents were pooled, resuspended in D5W, frozen at -80 °C without glycerol, and thawed one aliquot at a time for experiments. The utilization of adult donor cecal slurry to study neonatal sepsis could potentially transfer species of bacteria present in the cecal slurry that the neonatal mouse has not been exposed to, but it is a strategy that allows for the study of sepsis in neonatal mice and has been used to study neonatal mouse biology in the past13,14,15. Cecal slurry was diluted in D5W to provide nutrition to the bacteria, which allowed the establishment of an active infection once the bacteria were injected, and was done to mimic the availability of nutrients in the peritoneal cavity during necrotizing enterocolitis. Glycerol was not included as a stabilizing agent in freezing bacteria because of the potential negative side effects that could arise from glycerol injection alone. If glycerol had been included in the cecal slurry preparation, then the potential damage that glycerol alone could induce would need to be tested for by including a glycerol-only (lacking cecal slurry) injection in mice, which would have increased mouse usage. The bacteria viability of the cecal slurry stocks was tested after freezing the cecal slurry stock without glycerol and was found to be constant, with no change in bacteria concentration in separate aliquots of the same cecal slurry preparation stored at -80 °C over a 6 month period. This suggests that the storage without glycerol is feasible in providing a consistent biological outcome. The use of a bulk-prepared frozen cecal slurry stock also allowed for the use of mice bred in-house, reducing cost and utilizing male mice that would otherwise be excess from breeding, therefore reducing mouse wastage.
The identification of failed challenges in mice was important to avoid adding extra noise to the system. After undergoing an intraperitoneal injection of cecal slurry, the mice were observed for the presence of a bulge underneath the skin, which indicated a failed injection that was actually subcutaneous. Mice were observed for leaks at the injection site, both immediately after needle removal and after allowing them to take a step after the injection, because mice would sometimes (rarely) leak only after moving the limb of the injection site by taking a step. The presence of a bulge or leak following the injection resulted in removing the mouse from the analysis. After all, either of these could result in a different outcome due to the incorrect amount of cecal slurry injected as a 5% difference in challenge dose has been observed to affect subsequent survival.
Cecal slurry challenge experiments often required varying target lethal doses with varying weight-adjusted doses. Due to this, injection volumes can range from as little as 20 µL and up to 100 µL. The proportionate experimental error associated with dead needle volume also changes along with the injection volume, increasing the difficulty to directly compare different doses. With the simple modification of standardizing the injection volume, this source of variance is removed from the experiment.
The neonatal mouse's behavioral monitoring system used in this protocol is the first of its kind. Researchers intent on conducting ethical research with newborn mice are often faced with the challenging lack of resources to assess the animal's well-being at this age. The presented intuitive and consistent monitoring system begins to address this knowledge gap. Importantly, this evidence-driven approach not only increases the quality of the experimental data obtained but, at the same time, also reduces the suffering of the experimental animals.
Dr. James Wynn receives support from the National Institutes of Health (NIH)/National Institute of General Medical Sciences (R01GM128452) and the NIH/Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) (R01HD089939).
Special thanks to Claire Harrison and the Animal Care Facility at British Columbia Children's Hospital Research Institute (BCCHR) for their support in the animal work, as well as to Dr. Po-Yan Cheng for their guidance and input on animal monitoring and well-being.
|0.1 - 20 μL pipette tips
|1.8 mL Microcentrifuge tube
|100 - 1000 μL pipette tips
|1 - 200 μL pipette tips
|15 mL Centrifuge tube
|only the needle, not the syringe, used for pinning mouse to styrofoam
|28G 0.5 mL Insulin syringe
|2 mL Cryogenic vial
|50 mL Centrifuge tube
|5 mL pipette
|6 - 10 week old C57BL/6J adult mice
|7 + day old C57BL/6J neonatal mice
|Bred in house
|70 μm Cell strainer
|Defibrinated Sheep's Blood
|Dextrose 5% Water (D5W)
|Dissecting Scissors, Sharp Tip
|Dissecting Scissors, Sharp/Blunt Tip
|Ethanol (HistoPrep 95% Denatured Ethyl Alcohol)
|diluted to 70% with double distilled water
|Ethanol-proof marker; Lab marker
|EZ Anesthesia Vaporizer
|Germinator 500, Dry sterilize surgicial instrument (Hot bead sterilizer)
|Rainin Classic Pipette PR-1000
|Rainin Classic Pipette PR-20
|Rainin Classic Pipette PR-200
|BL 150 S
|82027-440 / 82027-442
|Square 1000 mL Storage Bottle
|Sure-Seal Mouse/Rat euthanasia chamber
|Tryptic Soy Agar
|VX-200 Lab Vortex Mixer
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