Research in the Kawakami lab was supported by NIH grants: R01 AR064418-01A1, R01 HL124283-01, R21 AI 115534-01, and R41AI124734-01.

All animal experiments were approved by the Animal Care and Use Committee of the La Jolla Institute for Allergy and Immunology.
1. Mouse Body Temperature Measurement During Anesthetization
<ol>
	<li>Place the mouse in an anesthesia induction box. Anesthetize by using 1 L/min flow of oxygen with 5% isoflurane. 
	NOTE: Anesthetization is confirmed when the mouse ceases voluntary movement and has been immobile for over 30 s. Alternatively, monitor respiratory rate, and once mice are breathing at 1 breath per every 2 s or longer, anesthetization is confirmed.</li>
	<li>Hold the mouse by the nape of the neck with an index finger and thumb and hold the tail with a pinky finger in order to expose the lower abdomen.</li>
	<li>Place the infrared thermometer sensor below the lower abdomen while holding the mouse with its body parallel to the ground. 
	NOTE: The outer flat surface of the thermometer (not the surface of the sensor) should be approximately 2&#8211;5 mm away from the surface of the abdomen. This replicate results of temperatures measured while mice are not anesthetized (described in section 2). It is important to determine the target site of the abdomen. Aiming between two upper nipples allows for consistent results.</li>
	<li>Hold the trigger to measure the temperature. Ensure the stable holding of the mouse and thermometer.</li>
</ol>
2. Mouse Body Temperature Measurement Without Anesthetic
<ol>
	<li>Pick up the mouse by the middle of the tail.</li>
	<li>Expose the abdomen of the mouse.
	<ol>
		<li>Allow the mouse to hold on to a straight-edge surface, such as the lip of an open cage or cage-top with its forepaws. 
		NOTE: This allows the mouse to stretch its upper body and expose the abdomen.</li>
		<li>Alternatively, allow the mouse to hold on to the upper straight-edge of the thermometer, and make the mouse sit on the outer flat surface of the thermometer with its abdomen just over the infrared sensor. 
		NOTE: Any time the hind paws are resting on the surface of the thermometer, ensure that the feet are not obstructing the sensor from the abdominal surface. Thermometers with foot obstruction will measure a temperature lower than that of the abdomen.</li>
	</ol>
	</li>
	<li>Hold the trigger to measure the temperature. 
	NOTE: Mice tend to move; take care in measuring the same location of the body consistently by taking temperature measurements when mice are relatively less mobile.</li>
</ol>
3. Passive Systemic Anaphylaxis7
<ol>
	<li>Day 0: Sensitize with immunoglobulin E (IgE).
	<ol>
		<li>Prepare 200 &#181;L (per mouse) of anti-dinitrophenyl (DNP) IgE at a concentration of 100 &#181;g/mL in PBS.</li>
		<li>Intraperitoneally inject 200 &#181;L of anti-dinitrophenyl (DNP) IgE prepared in step 3.1.1 or PBS only (PBS-only is the negative control). Use a 26 G needle for injection. Perform the injections just lateral to the midline, approximately between the two most inferior nipples.</li>
	</ol>
	</li>
	<li>Day 1: Induce anaphylaxis with DNP-HSA.
	<ol>
		<li>Prepare 100 &#181;L (per mouse) of DNP-HSA at a concentration of 10 mg/mL in 0.9% NaCl.</li>
		<li>Anesthetize the mice as described in step 1.1.</li>
		<li>Measure the body temperature using the technique described in step 1.</li>
		<li>Intravenously inject 100 &#181;L of DNP-HSA prepared in step 3.2.1. Use a 30 G needle for injection. Perform the injections on the retroorbital venous sinus. Insert the needle on the medial side of the eye at a shallow angle, aiming behind the eye.</li>
		<li>After injection, place the mice in individual cages. Ensure that the mice recover from anesthesia. Observe that they awaken and become voluntarily mobile.</li>
		<li>Measure body temperatures using the infrared thermometer and observe their activity every 10 min for 70 min.</li>
	</ol>
	</li>
</ol>
4. Mouse Model of Food Allergy8,9
NOTE: The schematic is shown in Figure 2.
<ol>
	<li>Day 0: Sensitize the mice with OVA/alum.
	<ol>
		<li>Prepare 100 &#181;L (per mouse) of OVA (0.5 mg/mL) and alum (10 mg/mL) together in PBS. Vortex on the low setting for 30 min.</li>
		<li>Anesthetize the mice as described in step 1.1.</li>
		<li>Using a 26 G needle, intraperitoneally inject each mouse with 100 &#181;L of the OVA/alum mixture prepared in step 4.1.1. 
		&#8203;NOTE: Vortex the OVA/alum mixture again, briefly prior to each injection, to best ensure a homogenized mixture. See step 3.1.3 for details on the injection site.</li>
		<li>After injection, place mice back in their original cages. Ensure that the mice recover from anesthesia. Observe that they awaken and become voluntarily mobile.</li>
	</ol>
	</li>
	<li>Day 14: Perform a second sensitization with OVA/alum.
	<ol>
		<li>Repeat steps 4.1.1, 4.1.2, and 4.1.3.</li>
	</ol>
	</li>
	<li>Days 28-46: Challenge the mice with OVA every other day.
	<ol>
		<li>Prepare 100 &#181;L (per mouse) of OVA at a concentration of 250 mg/mL in PBS. 
		&#8203;NOTE: The mixture should be made by gently rocking back and forth by hand rather than with a vortex to minimize bubble formation.
		<ol>
			<li>If using food allergy inhibitors, prepare the inhibitors9. Prepare 100 &#181;L (per mouse) of inhibitor at a concentration of 1 mg/mL in PBS. This allows a delivery of 100 &#181;g of inhibitor for each mouse. 
			&#8203;NOTE: Do not exceed 100 &#181;L of inhibitor per mouse, as the mice are only able to handle 200 &#181;L of total gavage volume during one challenge day. As 100 &#181;L are necessary for the OVA challenge as well, 100 &#181;L is the maximum recommended volume for inhibitor usage.</li>
		</ol>
		</li>
		<li>Anesthetize the mice as described in step 1.1.</li>
		<li>Orally gavage each mouse with 100 &#181;L of the OVA solution (25 mg of OVA in 100 &#181;L of PBS gavaged per mouse) that was prepared in step 4.3.1.
		<ol>
			<li>If using inhibitors, utilize the same techniques described in the following steps for the gavage of inhibitor (or PBS control) prepared in step 4.3.1.1 30 minutes prior to the OVA challenge. After 30 min, move on to the next step.</li>
			<li>Use a gavage needle with a 1 mL syringe. Take up 100 &#181;L of OVA into the syringe.</li>
			<li>Insert the needle into the mouth; then, pointing the needle toward the left or right side of the throat, gently slide the needle through the esophagus. Inject 100 &#181;L of OVA. 
			NOTE: To ensure that the needle is not in the trachea, observe active breathing before injecting the OVA. The insertion of the needle will also be resisted early if it has entered the trachea rather than the esophagus.</li>
		</ol>
		</li>
		<li>Place the mice into their individual cages. For easy observation of stool quality, use cages without bedding.</li>
		<li>Ensure recovery from anesthesia. Observe that the mice awaken and become voluntarily mobile.</li>
		<li>Measure body temperatures of the mice at timepoints 10, 20, 40, and 60 min using the technique described in section 2.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Finkelman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphylaxis:+lessons+from+mouse+models.">Anaphylaxis: lessons from mouse models.</a> The Journal of Allergy and Clinical Immunology. 120 (3), 506-515 (2007).</li><li>Lee, J. K., Vadas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphylaxis:+mechanisms+and+management.">Anaphylaxis: mechanisms and management.</a> Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology. 41 (7), 923-938 (2011).</li><li>Newsom, D. M., Bolgos, G. L., Colby, L., Nemzek, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+body+surface+temperature+measurement+and+conventional+methods+for+measuring+temperature+in+the+mouse.">Comparison of body surface temperature measurement and conventional methods for measuring temperature in the mouse.</a> Contemporary Topics in Laboratory Animal Science. 43 (5), 13-18 (2004).</li><li>Wong, J. P., Saravolac, E. G., Clement, J. G., Nagata, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+hypothermia+model+for+study+of+respiratory+tract+influenza+virus+infection.">Development of a murine hypothermia model for study of respiratory tract influenza virus infection.</a> Laboratory Animal Science. 47 (2), 143-147 (1997).</li><li>Quimby, J. M., Olea-Popelka, F., Lappin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+digital+rectal+and+microchip+transponder+thermometry+in+cats.">Comparison of digital rectal and microchip transponder thermometry in cats.</a> Journal of American Association for Laboratory Animal Science. 48 (4), 402-404 (2009).</li><li>Mei, J., Riedel, N., Grittner, U., Endres, M., Banneke, S., Emmrich, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature+measurement+in+mice+during+acute+illness:+implantable+temperature+transponder+versus+surface+infrared+thermometry.">Body temperature measurement in mice during acute illness: implantable temperature transponder versus surface infrared thermometry.</a> Scientific Reports. 8 (1), 3526 (2018).</li><li>Doyle, E., Trosien, J., Metz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+the+induction+and+evaluation+of+systemic+anaphylaxis+in+mice.">Protocols for the induction and evaluation of systemic anaphylaxis in mice.</a> Methods in Molecular Biology. 1032, 133-138 (2013).</li><li>Brandt, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+are+required+for+experimental+oral+allergen-induced+diarrhea.">Mast cells are required for experimental oral allergen-induced diarrhea.</a> The Journal of Clinical Investigations. 112 (11), 1666-1677 (2003).</li><li>Ando, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine-releasing+factor+enhances+food+allergy.">Histamine-releasing factor enhances food allergy.</a> The Journal of Clinical Investigations. 127 (12), 4541-4553 (2017).</li><li>Brandt, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+IL-4/IL-13+signaling+to+alleviate+oral+allergen-induced+diarrhea.">Targeting IL-4/IL-13 signaling to alleviate oral allergen-induced diarrhea.</a> The Journal of Allergy and Clinical Immunology. 123 (1), 53-58 (2009).</li></ol>

The authors have nothing to disclose.

The protocol described was established with the goal of measuring body temperature without the use of anesthesia. Despite its relative ease with which temperature readings can be obtained, there are several caveats that accommodate this technique, in addition to the more obvious effects such as handling stress and different ambient temperatures.
First, in order to maintain consistent temperature readings throughout the experiment, the location where the temperature is being measured must be predetermined using anatomic landmarks, and investigators must be able to replicate the measurement of said body location. By securing the mouse&#39;s forepaws on a straight-edge surface, such as the lip of a cage or the edge of the thermometer, the abdomen is readily exposed to the infrared sensor of the thermometer. Generally, the abdomen is a large surface area that allows some flexibility for consistent results. Furthermore, the abdomen has sparser hair than the dorsal surface of the mouse, and therefore allows for a more accurate body temperature reading.
Secondly, hind paws must always be monitored when taking a temperature measurement. When the thermometer is close to the abdomen, the mice tend to place their feet on the thermometer itself. When the feet or toes are within the circumference of the sensor, the temperature reading may be lower than that of the abdomen, as demonstrated in Video 4. It is important to allow the mouse to straddle with its feet on either side of the sensor, while keeping the circumference free of any body parts or objects besides the abdominal surface.
Thirdly, the abdominal surfaces of mice cannot be wet when taking the measurements. A wet surface will alter the thermometer&#39;s ability to measure temperature accurately; usually, the measured temperature will be lower than the actual value. This issue can be avoided by keeping the mice in cages with fresh bedding during the course of the observational period. This also ensures that the dryness of the abdominal surface is standardized each time the temperatures are measured.
Lastly, infrared thermometers have a larger margin of error than rectal probes, albeit only slightly. The margin of error for measurements with rectal probes is &#177; 0.1 &#176;C, whereas for infrared thermometers, it is &#177; 0.2 &#176;C. This margin of error was minimal compared to the overall differences in temperature drop between populations in the PSA experiment. More importantly, a project requiring a difference as subtle as &#177; 0.1 &#176;C may also foresee other margin of error issues, regardless of whether an infrared thermometer or rectal probe is used. During the preparation of this protocol, it had also been shown that there is a very good correlation between body core temperatures measured by implantable temperature transponders and body surface temperatures measured by infrared thermometers during lipopolysaccharide-induced hypothermia6.
Temperature drop in the food allergy model used had not been observed previously8,10. The standard for evaluation had previously been to determine the phenotype severity by diarrhea scoring; however, this method lacked objectivity. By using the infrared thermometer, we showed that subtle temperature drops could be detected. We were then able to correlate these with activity levels, as the mice were not anesthetized during the procedure. This method allows for a fast, accurate, easy-to-use, noninvasive, and inexpensive way to measure body temperature in mice while being able to observe other phenotypes that would otherwise be missed if mice are anesthetized&#160;for rectal probe measurements.

Measurement of body temperature has been an essential part of monitoring the effects of anaphylactic symptoms in animal models1,2. Temperature differences have been traditionally measured by rectal probe thermometers in mice3,4. With these measurements, investigators have reliably portrayed differences in temperature among variables; however, this method is a time-consuming procedure and causes distress to mice, which can increase the body core temperature. Rectal probing can also cause mucosal tearing and infection3. Moreover, the mice should be anesthetized in order to humanely insert the rectal probe to measure the temperature3. This is a slow process, and it prohibits the measurement of successive temperatures within a short period of time. Furthermore, mice activity phenotypes cannot be observed during this time until the anesthetic is completely worn off, which is another time-consuming process. More recently, other reliable methods to measure body temperature have used subcutaneously implanted passive infrared transponder tags or radio transmitters that include a temperature sensor3,5,6. Although they are accepted as the ideal practice by some researchers, these methods are not widely used because of high initial costs and distress to mice, due to the surgical implantation of a temperature sensor under the skin or another part of the body.
In order to demonstrate that a temperature difference is an accurate reflection of symptoms in a disease model1,2, mice must be awake during the temperature measurement and be able to return to their normal phenotypic activity immediately before and after the measurement. To this end, we sought a method by which this could be achieved.
Our goal was to accurately and inexpensively measure mouse body temperature, without the need for anesthesia and without restrictions on activity, to enable observation of behavioral phenotypes during and after the time of temperature measurement. To achieve this goal, it was apparent that a technique less invasive than the standard rectal temperature probes was required. Infrared thermometers have been used for decades in clinical medicine, especially in pediatrics, to obtain accurate temperature readings. It has been an alternative method that has allowed clinicians to quickly and accurately obtain temperature measurements in infants and fussy children that are actively mobile. We implemented this same technique in mice and have developed a successful method to obtain temperatures without anesthesia. Importantly, we show that this method is capable of replicating the well-established passive systemic anaphylaxis results regarding temperature changes, while also being able to observe the activity of the mouse throughout the measurement. Furthermore, we use the same method to evaluate body temperatures of food-allergic mice, while simultaneously investigating other symptoms, to demonstrate that body temperature is indeed an accurate reflection of the activity level and overall phenotype of the mouse.

<table border="0" cellpadding="0" cellspacing="0" style="width:744px;" width="744">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Non-contact infrared thermometer</td>
			<td style="width:164px;">SinoPie</td>
			<td style="width:127px;">DT-8861</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-dinitrophenyl (DNP) IgE</td>
			<td>Sigma Aldrich</td>
			<td>D8406-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrecisionGlide 30G needle</td>
			<td>BD</td>
			<td>305128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrecisionGlide 26G needle&#160;</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dinitrophenyl - human serum albumin</td>
			<td>Biosearch Technologies</td>
			<td>D-5059-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovalbumin from chicken egg white</td>
			<td>Sigma Aldrich</td>
			<td>A5503-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imject Alum</td>
			<td>ThermoFisher Scientific</td>
			<td>77161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animal Feeding Needles, disposable</td>
			<td>Fisher Scientific</td>
			<td>01-208-87</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse body temperature measurement using infrared thermometer during passive systemic anaphylaxis and food allergy evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Passive systemic anaphylaxis: For iv injection, 10 week old female BALB/c mice were anesthetized. Prior to the injection, we measured their body temperatures (Video 1) as described in step 1. Figure 1 shows the temperature trend of both populations after iv injection. The IgE-sensitized mouse showed a maximum temperature drop of 3.0 &#176;C at 20 min, while the PBS control mouse had a maximum drop of 1.1 &#176;C at 20 min7. Furthermore, throughout this process, activity scores were evaluated based on the mobility of each mouse. Mice were first observed without any agitation. Then, they were observed with agitation by introducing the evaluator&#39;s hand into the cage and making soft contact with the mouse. Based on their reactions, the mice are evaluated and scored on a scale of 1 to 3. A score of 1 indicates a mouse that is immobile or minimally mobile with and without agitation; a score of 2 indicates a mouse that is immobile without agitation and is mobile with agitation without making quick evasive movements; and a score of 3 indicates a mouse that is either mobile or immobile without agitation, and mobile with agitation and makes quick evasive movements. Videos 2 and 3 provide examples of the two groups that exemplify the temperature measurement methods as well as the corresponding activity level measurements. Usage of the infrared thermometer allowed for temperature measurements without anesthesia, which in turn allowed for behavioral phenotype evaluation before, during, and after the temperature measurement; in contrast, a traditional rectal probe would require anesthesia and therefore preclude any behavioral evaluation within the several minutes surrounding the temperature measurements.
Mouse model of food allergy8: The schematic of the mouse model is shown in Figure 2.&#160;Mice are female BALB/c mice of ages 8 to 10 weeks old at the time of sensitization. Mice were observed at 10, 20, 40, and 60 min after each challenge for body temperature and activity. A representative result is shown in Figure 3 of the temperature change during challenge 7. The mice showed a maximal temperature drop at 10 min; therefore, the 10 min temperatures were used to correlate with body temperatures at the 10 min activity levels (with scoring based on the criteria depicted above). Activity score and temperature drop showed a statistically significant correlation (Figure 4). As mentioned in the passive systemic anaphylaxis section, behavioral observations were performed due to the lack of anesthesia, and the infrared thermometer was utilized. In contrast, rectal probes would forestall any behavioral observations or evaluations during the time of temperature measurement. Any time that temperature and other activity phenotypes must be recorded, it is essential to utilize a temperature-measuring system that does not require anesthesia.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58391/58391fig1.jpg" /> 
Figure 1: Passive systemic anaphylaxis in BALB/c mice. Two mice were ip injected with anti-DNP IgE on day 0, then iv injected with either normal saline or DNP-HSA on day 1. Body temperature was measured using an infrared thermometer before injection and every 10 min after injection for 70 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58391/58391fig2.jpg" /> 
Figure 2: Food allergy mouse model schematic. On day 0, BALB/c mice were sensitized ip with OVA and alum, and again on day 14. On day 28, 30 min prior to each challenge, mice were orally gavaged with either PBS control or a food allergy inhibitor (HRF-2CArecombinant histamine-releasing factor with its two cysteines exchanged with alanines)9. Mice were then challenged ig with 25 mg of OVA. Challenges were performed every other day for 9 challenges. Mice were observed for body temperature measurement and activity scoring at 10, 20, 40, and 60 min after the challenge. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58391/58391fig3.jpg" /> 
Figure 3: Food allergy model in BALB/c mice. Temperature change after the 7th OVA challenge was monitored by infrared thermometer. Typical results are shown from at least four independent experiments (**, p &#60;0.01 between PBS and 2CA treatments by Student&#39;s t-test; ns: not significant). Error bars = SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58391/58391fig4.jpg" /> 
Figure 4: Correlation between activity and temperature at 10 min in food allergy experiments. The results reflect measurements taken from 4 separate sets of experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie Image 1" src="/files/ftp_upload/58391/58391movie1.jpg" /> 
Video 1: Body temperature measurement while mice are anesthetized. Mice body temperature measurements can be taken with or without anesthesia. For consistent measurements, it is essential to always aim the thermometer at the same body location. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2416.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie Image 2" src="/files/ftp_upload/58391/58391movie2.jpg" /> 
Video 2: Activity level at 40 min after DNP-HSA iv injection. Mice are observed without agitation initially, then with agitation. Based on their reactions, the mice were evaluated and scored on an activity scale of 1 to 3. The mouse on the left (anti-DNP sensitized mouse) is given a score of 1, while the mouse on the right (PBS control mouse) is given a score of 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2442.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie Image 3" src="/files/ftp_upload/58391/58391movie3.jpg" /> 
Video 3: Body temperature measurement at 40 min after DNP-HSA iv injection. This figure demonstrates the ability of the infrared thermometer to accurately and noninvasively measure body temperature without anesthetization. Moreover, the activity scores demonstrated in Video 2 reflect the degree of temperature drop measured in this video. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2446.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie Image 4" src="/files/ftp_upload/58391/58391movie4.jpg" /> 
Video 4: Errors in temperature reading by hind paw obstruction of the sensor. This video demonstrates the difference in temperatures with and without the foot within sensor circumference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2455.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation at JoVE.com

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

mouse-body-temperature-measurement-using-infrared-thermometer-during

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

25.3K Views.  La Jolla Institute for Allergy and Immunology. Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Video: Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by ¹H NMR Spectroscopy

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Measuring Local Anaphylaxis in Mice

Detection of True IgE-expressing Mouse B Lineage Cells

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

Evaluation of Extracellular Vesicle Function During Malaria Infection