N.G. is the recipient of fellowships from the French &#8220;Fondation pour la Recherche M&#233;dicale FRM&#8221; and the Philipp Foundation; R.S. is supported by the Lucile Packard Foundation for Children&#8217;s Health and the Stanford NIH/NCRR CTSA award number UL1 RR025744; P.S. is supported by a Max Kade Fellowship of the Max Kade Foundation and the Austrian Academy of Sciences and a Schroedinger Fellowship of the Austrian Science Fund (FWF): J3399-B21; S.J.G. acknowledges support from National Institutes of Health grants U19 AI104209, NS 080062 and from Tobacco-Related Disease Research Program at University of California; L.L.R. acknowledges support from the Arthritis National Research Foundation (ANRF) and National Institutes of Health grant K99AI110645.

All animal care and experimentation were conducted in compliance with the guidelines of the National Institutes of Health and with the specific approval of the Institutional Animal Care and Use Committee of Stanford University.
1. Generation and Characterization of Bone Marrow-derived Cultured Mast Cells (BMCMCs).
Note: Donor BMCMCs should be generated from bone marrow cells of the same genetic background as the recipient MC-deficient mice. Male-derived donor BMCMCs are not suitable for engraftment of female mice. Female-derived donor BMCMCs will successfully engraft into both male and female recipients.
<ol>
	<li>Bone Marrow Extraction.
	<ol>
		<li>Pour sterile phosphate-buffered saline (PBS) into 6 well culture plate (1 well per mouse) and place on ice.</li>
		<li>Soak the dissection instruments in 70% ethanol for sterilization.</li>
		<li>Euthanize donor mice by carbon dioxide (CO2) inhalation followed by cervical dislocation, then spray the mice with 70% ethanol at the sites of manipulation.</li>
		<li>Use sterile dissection instruments to extract the femur, tibia and fibula bones without cutting any bone epiphyses.</li>
		<li>While securing the bones with a forceps, scrape off all tissue from bones (and discard fibula) using sterile scissors (or scalpel blades). Place bones in PBS on ice until all bones are collected.</li>
		<li>Carry out all remaining steps in sterile tissue culture hood. Using sterile instruments, secure bones with forceps and cut off both epiphyses to expose the medullary cavity.</li>
		<li>Using 3 ml syringes and 30 G needles, and cold flushing medium (Dulbecco Modified Eagle Medium [DMEM] supplemented with 10% fetal calf serum [FCS, heat-inactivated], 2 mM L-glutamine, 1% antibiotic-antimycotic solution, 50 &#181;M &#914;-mercaptoethanol), flush the red bone marrow into a Petri dish.</li>
		<li>Use the same syringe to dissociate flushed bone marrow cell clusters by repeated gentle aspiration and ejection.</li>
		<li>Pool femoral and tibial bone marrow from each mouse into one 15 ml centrifuge tube. Fill the tube with cold flushing medium to wash bone marrow cells and centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>BMCMCs Culture.
	<ol>
		<li>Remove supernatant and recover pelleted bone marrow cells in 10 ml of culture medium (DMEM supplemented with 10% fetal calf serum [FCS, heat-inactivated], 2 mM L-glutamine, 1% antibiotic-antimycotic solution, 50 &#181;M &#914;-mercaptoethanol and 20% WEHI-3 cell-conditioned medium [as a source of IL-3; alternatively use recombinant mouse IL-3 at 10 ng/ml]).</li>
		<li>Place cells in tissue culture flask of appropriate size (e.g., T25 for 1 mouse, T75 for 2 mice, etc.).</li>
		<li>1-2 days after plating cells, transfer medium and suspension cells (leaving debris and adherent cells sticking to bottom of flask) to a new flask and add fresh culture medium (10 ml/mouse).</li>
		<li>During the following weeks, feed cells every 3-4 days (add 10 ml of culture medium per mouse). Maintain cell density between 2.5 x 105-1 x 106 cells/ml. Transfer non-adherent cells to a new flask once a week until no adherent cells are present in the culture flask. Test maturity of cells (see below) before use for in vitro assays or engraftment into MC-deficient mice. 
		NOTE: Complete differentiation of BMCMCs will take 4-6 weeks.</li>
	</ol>
	</li>
	<li>Assessment of BMCMC maturity.
	<ol>
		<li>Using flow cytometry.
		<ol>
			<li>Wash cells (5 x 104-5 x 105 cells/condition) with ice-cold FACS buffer (PBS, 0.5% FCS) in 5 ml polystyrene round bottom tube. Centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate supernatant.</li>
			<li>Dilute anti-mouse CD16/32 (clone 93 or 2.4G2) monoclonal antibodies 1:200 (2.5 &#956;g/ml) in FACS buffer. Add 10 &#181;l to each pellet and resuspend by brief vortexing. Incubate 5 min on ice to block Fc binding. 
			NOTE: Protect samples from direct light for all remaining steps.</li>
			<li>Dilute phycoerythrin (PE) conjugated anti-mouse Fc &#949;RI &#945; (clone MAR-1) 1:200 (1 &#956;g/ml) and fluorescein isothiocyanate (FITC) conjugated anti-mouse KIT (CD117; clone 2B8) 1:200 (2.5 &#956;g/ml) (&#8220;staining solution&#8221;), or the respective labeled isotype control antibodies in FACS buffer (&#8220;isotype control solution&#8221;).</li>
			<li>Split half of the blocked cells from step 1.3.1.2) into an additional polystyrene round bottom tube and add 20 &#181;l of &#8220;staining solution&#8221; in one tube and 20 &#181;l of &#8220;isotype controls solution&#8221; in the other tube. Incubate 30 min on ice.</li>
			<li>Add 3 ml ice-cold FACS buffer and centrifuge at 400 x g for 5 min at 4 &#176;C. Discard supernatant and resuspend the pellet in 300 &#181;l FACS buffer containing 1 &#181;g/ml propidium iodide (PI, for identification of dead cells). Proceed to flow cytometry analysis (see Figure 2A).</li>
		</ol>
		</li>
		<li>Using toluidine blue staining.
		<ol>
			<li>Wash 1 x 105 cells once with PBS by centrifugation at 400 x g for 5 min at room temperature, then aspirate and resuspend cells in 200 &#181;l of PBS.</li>
			<li>Transfer the cell suspension into a prepared cytofunnel attached to a microscope slide and proceed with cyto-centrifugation using a cytocentrifuge (40 x g for 5 min).</li>
			<li>Air dry slides for 10 min and draw a wax circle around cells using a PAP pen. Add 0.1% Toluidine blue solution to cover the cells. Incubate for 1 min. Wash slides in running tap water for 1 min, then air-dry slides for 10 min.</li>
			<li>Coverslip cells using mounting medium. Proceed to light microscope analysis (see Figure 2B)</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Engraftment of Mast Cell-deficient Mice with BMCMCs.
<ol>
	<li>Ear engraftment by intradermal (i.d.) injection. 
	NOTE: For adoptive transfer of BMCMCs into the ear pinnae of MC-deficient mice, we recommend performing two injections of 1 x 106 cells each (for a total of 2 x 106 cells) per ear pinna. Intradermal engraftment of BMCMCs into the ear pinnae of MC-deficient mice has been used by many investigators in several models, including models of passive cutaneous anaphylaxis (PCA)24,27, host defense against venoms28, and chronic hypersensitivity (CHS) reactions29,30.
	<ol>
		<li>Count and resuspend cells at 4 x 107 BMCMCs/ml (1 x 106 BMCMCs/ 25 &#181;l) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 30 G needle. Keep on ice until injection.</li>
		<li>Anesthetize 4-6 weeks old MC-deficient mice using isoflurane (2.5% v/v). Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Apply ophthalmic ointment with a Q-tip to prevent dryness of eyes.</li>
		<li>With the index finger, create vertical pressure on the dorsal face of the ear pinna to expose and stretch the ventral face.</li>
		<li>Perform two 25 &#181;l i.d. injections of BMCMC solution into two different sites of the ventral face of the ear pinna, the first injection in the middle of the ear and the second injection toward the tip of the ear pinna. Wait 4-6 weeks after i.d. engraftment before performing in vivo experiments. 
		Note: In our experience, by that time the number of MCs/mm2 of dermis in the central part of the ear pinna generally is similar to that in the corresponding location in wild type mice, whereas the numbers of MCs/mm2 in the periphery of the ear pinnae are typically substantially lower than those in the corresponding wild type mice27,28. This should be kept in mind when designing experiments with such MC-engrafted mice (e.g., agents with possible effects on MC function should be injected into the central part of the ear pinnae, and the analysis of the effects of such treatments should also focus on that area).</li>
	</ol>
	</li>
	<li>Peritoneal cavity engraftment by intraperitoneal (i.p.) injection. 
	NOTE: Adoptive transfer of BMCMCs into the peritoneal cavity of MC-deficient mice will require one injection of 2 x 106 cells per mouse. Such i.p. injections of BMCMCs into MC-deficient mice have been used by many groups to study the roles of peritoneal MCs in various models, including models of host defense against venoms31 or bacterial sepsis32,33.
	<ol>
		<li>Count appropriate number of cells and resuspend at 1 x 107 BMCMCs/ml (2 x 106 BMCMCs/ 200 &#181;l) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 25 G needle. Keep on ice until injection.</li>
		<li>Perform 1 injection of 200 &#181;l BMCMCs solution into the peritoneal cavity of 4-6 week old MC-deficient mice. Wait 4-6 weeks after i.p. engraftment before performing in vivo experiments.</li>
	</ol>
	</li>
	<li>Engraftment by intravenous (i.v.) injection. 
	NOTE:Adoptive transfer of BMCMCs by i.v. injection into MC-deficient mice will require one injection of 5 x 106 cells per mouse. Intravenous (i.v.) injections of BMCMCs into MC-deficient mice have been used by many groups to study the roles of MCs in various disease models, including models of bladder infection34, asthma35, lung fibrosis36, and antibody-mediated arthritis37.
	<ol>
		<li>Count appropriate number of cells and resuspend at 2.5 x 107 BMCMCs/ml (5 x 106 BMCMCs/200 &#181;l) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 30 G needle. Keep on ice until injection.</li>
		<li>Anesthetize 4-6 weeks old MC-deficient mice using isoflurane (2.5% v/v). Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Apply ophthalmic ointment with a Q-tip to prevent dryness of eyes.</li>
		<li>Perform one injection of 200 &#181;l of BMCMC solution into the tail vein (or, alternatively, the retro-orbital vein) of a MC-deficient mouse. Wait 12 weeks after i.v. engraftment before performing in vivo experiments.</li>
	</ol>
	</li>
</ol>
3. Analysis of Engrafted Mast Cell-deficient Mice.
<ol>
	<li>Ear engraftment analysis.
	<ol>
		<li>At the end of the experiment, euthanize mice by CO2 inhalation followed by cervical dislocation.</li>
		<li>Isolate the ear pinnae and fix in 10% (vol/vol) buffered formalin overnight at 4 &#176;C. Embed fixed ear pinnae in paraffin, cut 4 &#181;m sections of ear pinnae and mount on glass slides.</li>
		<li>Stain the slides with a 0.1% Toluidine blue solution for 1 min at room temperature. Wash slides in tap water for 1 min, then air-dry slides for 10 min at room temperature.</li>
		<li>Coverslip slides using mounting medium, then count the number of engrafted MCs per pinnae sections using a light microscope. Evaluate the engraftment efficiency by comparing the percentage and distribution of MCs in the ear skin of engrafted mice versus wild type mice.</li>
	</ol>
	</li>
	<li>Peritoneal cavity engraftment analysis.
	<ol>
		<li>Evaluation of mast cells number in the peritoneal cavity.
		<ol>
			<li>At the end of the experiment, euthanize mice by CO2 inhalation followed by cervical dislocation. Remove carefully the ventral skin of the mice without breaking the peritoneal cavity.</li>
			<li>Inject 5 ml of cold or room temperature PBS in the peritoneal cavity using a 5 ml syringe equipped with a 25 G needle. Use cold PBS to reduce the risk of activating peritoneal cells (this is very important when evaluating peritoneal MC degranulation or levels of some MC-derived products in the peritoneal lavage fluid). Perform a massage of the abdomen for 20 sec to harvest peritoneal cells.</li>
			<li>Slowly aspirate the peritoneal lavage using a 5 ml syringe equipped with a 22 G needle. Record the volume of aspirated lavage (expect to recover up to 80% of injected volume).</li>
			<li>Transfer peritoneal lavage fluid into a 5 ml polystyrene round bottom tube and centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate supernatant. Recover the pellet in 400 &#181;l cold PBS.</li>
			<li>Count total number of peritoneal cells using a hemocytometer chamber. Use half of the cell suspension for a flow cytometry analysis (as described in step 1.3.1), to evaluate the percentage and marker expression of engrafted MCs in the peritoneal cavity. Multiply the total number of peritoneal cells by the percentage of MCs to obtain absolute number of peritoneal MCs.</li>
			<li>Perform a cytocentrifugation with the remaining cells as described in step 1.3.2.2. Air dry slides for 10 min at room temperature and draw a wax circle around cells using a PAP pen.</li>
			<li>Cover cells with undiluted May-Gr&#252;nwald Staining solution for 5 min, followed by washing in PBS for 5 min, both at room temperature. Cover cells with Giemsa stain diluted 1:20 with deionized water and incubate 20 min at room temperature. Wash slides 2 times in tap water for 1 min, then air-dry slides.</li>
			<li>Coverslip cells using mounting medium, and calculate the percentage of engrafted MCs using a light microscope (by counting at least 400 total cells). Evaluate the engraftment efficiency by comparing the percentage of MCs in the peritoneal lavage of engrafted mice versus wild type mice.</li>
		</ol>
		</li>
		<li>Evaluation of mast cells in the mesenteric windows.
		<ol>
			<li>Following the peritoneal lavage described in step 3.2.1, cut open the peritoneal membrane to expose the intestinal tract of the mice. Arrange 4 to 5 mesenteric windows per mouse onto a slide.</li>
			<li>Fix the slides for 1 hr in Carnoy solution (3:2:1 vol/vol/vol of ethanol, chloroform, and glacial acetic acid) at room temperature. Air-dry slides and remove the intestine (the fixed mesenteric windows will remain attached to the slides).</li>
			<li>Stain the preparations for 20 min at room temperature with Csaba (Alcian blue/Safranin O) staining solution.
			<ol>
				<li>To make 500 ml of Csaba stain, prepare 500 ml of acetate buffer by mixing 100 ml of 1 M sodium acetate solution with 120 ml of 1 M HCl. Make up to 500 ml with deionized water and adjust pH to 1.42. Then, dissolve 90 mg Safranin [identifies &#8216;mature MCs&#8217; in red], 1.8 g Alcian blue [identifies &#8216;immature MCs&#8217; in blue] and 2.4 g ferric ammonium sulphate NH4Fe(SO4)2 into 500 ml of acetate buffer.</li>
			</ol>
			</li>
			<li>Coverslip slides using mounting medium, then count the number of engrafted MCs per mesenteric window using a light microscope. Evaluate the engraftment efficiency by comparing the percentage and distribution of MCs in the mesenteric windows of engrafted mice versus wild type mice.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>I.v. engraftment analysis.
	<ol>
		<li>At the end of the experiment, euthanize mice by CO2 inhalation followed by cervical dislocation, and harvest tissue of interest (e.g. lung, skin or spleen) and fix overnight in 10% (vol/vol) buffered formalin at 4 &#176;C.</li>
		<li>Embed fixed tissues in paraffin, cut 4 &#181;m sections, and mount on glass slides. Stain the slides with a 0.1% Toluidine blue solution for 1 min at room temperature. Wash slides in tap water for 1 min, then air-dry slides for 10 min.</li>
		<li>Coverslip slides using mounting medium and evaluate number and distribution of engrafted MCs per tissue sections using a light microscope.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Almost 30 years after its initial description38, the &#8216;mast cell knock-in&#8217; approach continues to provide valuable information about what MCs can do or can&#8217;t do in vivo. The functions of MCs were long thought to be limited to their role in allergy. Data generated using the &#8216;mast cell knock-in&#8217; approach have changed this view, by providing evidence that MCs can, among other functions, play critical roles in host defense against certain pathogens4,39</s...

Mast cells (MCs) are hematopoietic cells that arise from pluripotent bone marrow progenitors1-3. Following bone marrow egression, MCs progenitors migrate into various tissues where they develop into mature MCs under the influence of local growth factors1-3. Tissue-resident MCs are strategically located at host-environment interfaces, such as the skin, the airways and the gastrointestinal tract, where they behave as a first line of defense against external insults3-6. MCs are often sub-classified based on their &#8220;baseline&#8221; phenotypic characteristics and their anatomic locations. In mice, two types of MCs have been described: &#8220;connective tissue-type&#8221; MCs (CTMCs) and mucosal MCs (MMCs)1-3,7,8. CTMCs are often located around venules and near nerve fibers, and reside in serosal cavities, while MMCs occupy intraepithelial locations in the gut and respiratory mucosa1-3.
Numerous methodologies have been applied to study biological functions of MCs9-13. Many groups have focused on in vitro approaches using either cell lines (such as the human MC lines HMC114 or LAD215,16), in vitro derived MCs (such as human peripheral blood-derived MCs17, or mouse bone marrow-derived cultured MCs [BMCMCs]18, fetal skin-derived cultured MCs [FSCMCs]19 and peritoneal cell-derived MCs [PCMCs]20) or ex vivo isolated MCs from different anatomical sites. All these models are widely used to study molecular details of MC biology, such as signaling pathways involved in MC activation. However, an important aspect of MCs biology is that their phenotypic and functional characteristics (e.g., cytoplasmic granule protease content or response to different stimuli) can be modulated by anatomical location and microenvironment2,7. Since the exact mixture of such factors that are encountered in vivo may be difficult to reproduce in vitro, we favor using in vivo approaches to gain insights into MCs functions9.
Several mouse strains with genetic MC deficiency exist, such as the widely used WBB6F1-Kit W/W-v or C57BL/6-Kit W-sh/W-sh mice. These mice lack expression and/or activity of KIT (CD117), the receptor for the main MC growth factor stem cell factor (SCF)21,22. As a result, these mice have a profound MC deficiency but also have additional phenotypic abnormalities related to their c-kit mutations (in the WBB6F1-Kit W/W-v mice) or to the effects of the large chromosomal inversion that results in reduced c-kit expression (in the C57BL/6-Kit W-sh/W-sh mice)9,10,12,23. More recently, several strains of mice with c-kit-independent constitutive MC deficiency have been reported24-26. All these mice and some additional new types of inducible MC-deficient mice have been recently reviewed in detail9,10,13.
Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This so-called &#8216;mast cell knock-in&#8217; method can be used to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

<table><tbody><tr><td>1% Antibiotic-Antimycotic Solution</td><td>Corning cellgro</td><td>30-004-Cl</td><td></td></tr><tr><td>3 ml Syringe</td><td>Falcon</td><td>309656</td><td></td></tr><tr><td>35 mm x 10 mm Dish</td><td>Corning cellgro</td><td>430588</td><td></td></tr><tr><td>5 ml Polystyrene Round Bottom Tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>Acetic Acid Glacial</td><td>Fisher Scientific</td><td>A35-500</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>Rowley Biochemical Danver</td><td>33864-99-2</td><td></td></tr><tr><td>Allegra 6R Centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Anti-mouse CD16/32 (clone 93) Purified</td><td>eBioscience</td><td>14-0161-81</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M7522</td><td></td></tr><tr><td>BD 1 ml TB Syringe</td><td>BD Syringe</td><td>309659</td><td></td></tr><tr><td>BD 22 G x 1 (0.7 mm x 25 mm) Needles</td><td>BD Precision Glide Needle</td><td>205155</td><td></td></tr><tr><td>BD 25 G 5/8 Needles</td><td>BD Syringe</td><td>305122</td><td></td></tr><tr><td>BD 30 G x 1/2 Needles</td><td>BD Precision Glide</td><td>305106</td><td></td></tr><tr><td>Blue MAX Jr, 15 ml Polypropylene Conical Tube</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C298-500</td><td></td></tr><tr><td>Cytoseal 60 Mounting Medium</td><td>Richard-Allan Scientific</td><td>8310-4</td><td></td></tr><tr><td>Cytospin3</td><td>Shandon</td><td>NA</td><td></td></tr><tr><td>DakoCytomation pen</td><td>Dako</td><td>S2002</td><td></td></tr><tr><td>Dulbecco Modified Eagle Medium (DMEM) 1x</td><td>Corning cellgro</td><td>15-013-CM</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E 7023-500ml</td><td></td></tr><tr><td>Fetal Bovine Serum Heat Inactivated</td><td>Sigma Aldrich</td><td>F4135-500ml</td><td></td></tr><tr><td>FITC Conjugated IgG2b K Rat Isotype Control</td><td>eBioscience</td><td>14-4031-82</td><td></td></tr><tr><td>Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)</td><td>eBioscience</td><td>11-1171-82</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher Scientific</td><td>F79-500</td><td></td></tr><tr><td>Giemsa Stain Modified</td><td>Sigma Aldrich</td><td>GS-1L</td><td></td></tr><tr><td>Isothesia</td><td>Henry Schein Animal Health</td><td>29405</td><td></td></tr><tr><td>May-Grunwald Stain</td><td>Sigma Aldrich</td><td>MG-1L</td><td></td></tr><tr><td>Multiwell 6 well plates</td><td>Falcon</td><td>35 3046</td><td></td></tr><tr><td>Olympus BX60 Microscope</td><td>Olympus</td><td>NA</td><td></td></tr><tr><td>Paraplast Plus Tissue Embedding Medium</td><td>Fisher Brand</td><td>23-021-400</td><td></td></tr><tr><td>PE Conjugated IgG Armenian Hamster Isotype Control</td><td>eBioscience</td><td>12-4888-81</td><td></td></tr><tr><td>Phosphate-Buffered-Saline (PBS) 1x</td><td>Corning cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)</td><td>eBioscience</td><td>12-5898-82</td><td></td></tr><tr><td>Propidium Iodide Staining Solution</td><td>eBioscience</td><td>00-6990-50</td><td></td></tr><tr><td>Recombinant Mouse IL-3</td><td>Peprotech</td><td>213-13</td><td></td></tr><tr><td>Safranin-o Certified</td><td>Sigma Aldrich</td><td>S8884</td><td></td></tr><tr><td>Tissue culture flasks T25 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Beckton Dickinson</td><td>353109</td><td></td></tr><tr><td>Tissue culture flasks T75 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Beckton Dickinson</td><td>353110</td><td></td></tr><tr><td>Toluidine Blue 1% Aqueous</td><td>LabChem-Inc</td><td>LC26165-2</td><td></td></tr><tr><td>Recombinant Mouse SCF</td><td>Peprotech</td><td>250-03</td><td></td></tr></tbody></table>

analyzing the functions of mast cells in vivo using 'mast cell knock-in' mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the &#8216;mast cell knock-in&#8217; approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

An overview of the &#8216;mast cell knock-in&#8217; approach is shown in Figure 1, and includes the generation of BMCMCs, the number of cells that should be engrafted i.p., i.d. or i.v. into MC-deficient mice (the number can be varied if indicated based on the experimental design) and the interval between engraftment and experiment depending on the injection site (this interval also can vary, if indicated; e.g., the content of stored mediators in MC cytoplasmic granules increases steadi...

Watch this Scientific Journal Video about Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice at JoVE.com

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

analyzing-functions-mast-cells-vivo-using-mast-cell-knock

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Immunology and Infection

10.3K Views.  Stanford University School of Medicine. We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

Video: Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

Medicine

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Developmental Biology

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Summary

Explore More Videos

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca²⁺-imaging and Degranulation Assays

An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice