an in vitro method to identify innate and adaptive immune cells residing in murine lungs

An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

Transfer the lung to a Petri dish. Mince it using two fine scalpels, and then place it into a 50-milliliter conical tube. Add 5 milliliters of digestion buffer to wash the plate. Secure the lid of the tube, and digest the lung for 30 minutes on an orbital shaker at a speed of 150 rotations per minute at 37 degrees Celsius. Stop the reaction by adding 10 milliliters of cold BSA buffer.
After digestion, mix and dissolve the lung pieces using an 18-gauge needle. Place a 70-micrometer filter strainer on top of a new 50-milliliter conical tube, and transfer the digested lung mixture into the strainer.
Use the rubber side of a 10-milliliter syringe plunger to smash the remaining lung pieces on the filter, and wash it with BSA buffer. Centrifuge the single-cell suspension at 350 g for 8 minutes at 7 degrees Celsius. Discard the supernatant, and resuspend the cells in 1 milliliter of ACK lysis buffer. Mix the suspension using a 1-milliliter pipette, and incubate it for 90 seconds at room temperature.
Add 10 milliliters of cold BSA buffer to the reaction mixture, and centrifuge it at 350 g for 7 minutes at 4 degrees Celsius. Discard the supernatant. Resuspend the pellet in staining buffer, and count the cells using a hemocytometer. Resuspend the cells at a concentration of 5 million cells per milliliter for surface staining.
Transfer 1 million cells in 200 microliters per well into a 96-well plate, and centrifuge the plate at 350 g for 7 minutes at 4 degrees Celsius. Prepare a flow cytometry block solution by diluting anti-16/32 antibody in staining buffer.
Resuspend the cells in 50 microliters of the flow cytometry block solution. Incubate the suspension for 15 to 20 minutes at 4 degrees Celsius, or on ice. Then, add 150 microliters of staining buffer to the plate, and centrifuge it at 350 g for 5 minutes at 4 degrees Celsius.
Prepare surface antibody solution by diluting the surface antibodies in staining buffer. Resuspend the cells in 50 microliters of the surface antibody solution, and incubate the plate at 4 degrees Celsius in the dark. Then, wash the cells twice with staining buffer.
Prepare the fixation and permeabilization buffer by mixing three parts fixation and permeabilization diluent, and one part staining buffer. Resuspend the cells in 50 microliters of the preprepared buffer per well of the 96-well plate, and incubate them for 20 to 25 minutes at 4 degrees Celsius in the dark.
Dilute the permeabilization buffer with purified deionized water to prepare one times permeabilization buffer, and use it to wash the cells. Prepare an intracellular antibody solution by diluting with 1 milliliter of permeabilization buffer. Resuspend the cells in 50 microliters of the diluted intracellular antibody solution per well of the 96-well plate, and incubate for 40 minutes at 4 degrees Celsius in the dark.
Wash the cells with permeabilization buffer, then with staining buffer. After the final wash, resuspend the cells in 200 microliters of staining buffer. Acquire a minimum of 1.5 million cells per sample on the flow cytometer.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Surgical excision and tissue preparation
<ol>
	<li>Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol;&#160;Table of Materials). 
	NOTE: CO2&#160;asphyxiation should be avoided in lung studies as it might cause lung injury and alter the features and properties of lung immune cells. Cervical dislocation should also be avoided as it might cause mechanical injury of the lung.</li>
	<li>Transfer the mouse to a clean and dedicated area for surgical operation.</li>
	<li>Stabilize the mouse dorsal side down by using needles or tape on the four extremities. Use 70% ethanol to sanitize the skin of the ventral area.</li>
	<li>Perform an incision in the skin, from the neck to the abdomen. Carefully remove the skin from the thoracic area.</li>
	<li>Carefully remove the sternum and ribs.</li>
	<li>Flush the lungs by injecting 10 mL of cold phosphate-buffered saline (PBS) directly in the right ventricle, using an 18-21 G needle, until the lungs become completely white.</li>
	<li>Carefully remove the thymus and heart without touching the lungs.</li>
	<li>Gently detach the lungs from the surrounding tissues and transfer them to a tube with cold bovine serum albumin (BSA) buffer (Table 1). 
	NOTE: Effort should be made to remove all adjacent fat from the lungs before further preparing the single-cell suspension, as this could bias the readouts.</li>
</ol>
2. Preparation of single-cell suspension
<ol>
	<li>Transfer the lungs to an empty Petri dish and mince them with two fine scalpels. Transfer all the pieces of the minced lung to a new 50 mL conical tube. Use 5 mL of digestion buffer to wash the plate and add it to the 50 mL tube containing the minced lung (Table 1). 
	NOTE: Digestion buffer should be prepared immediately before use. Use 5 mg/mL of collagenase. Combining 1 or 5 mg of collagenase with BSA buffer or protein-free PBS did not improve results (Figure 1).</li>
	<li>Secure the lid of the tube and digest the lung for 30 min on an orbital shaker at a speed of 150 rpm at 37 &#176;C. Stop the reaction by adding 10 mL of cold BSA buffer.</li>
	<li>After digestion, use an 18 G needle to mix and dissolve the lung pieces. Place a 70 &#181;m filter strainer at the top of a new 50 mL conical tube. 
	NOTE: Usage of a smaller micron filter might result in the loss of major myeloid populations.</li>
	<li>Slowly transfer the digested lung mixture directly on the strainer. Use the rubber side of a 10 mL syringe plunger to smash the remaining lung pieces on the filter. Wash the processed material on the filter with BSA buffer.</li>
	<li>Centrifuge the single-cell suspension at 350 &#215; g for 8 min at 4 &#176;C.</li>
	<li>Carefully discard the supernatant and resuspend the cells in 1 mL of ammonium&#8211;chloride&#8211;potassium (ACK) lysis buffer. Mix well using a 1 mL pipet, and incubate for 90 s at room temperature.</li>
	<li>Add 10 mL of cold BSA buffer to stop the reaction and centrifuge at 350 &#215; g for 7 min at 4 &#176;C.Carefully discard the supernatant and resuspend the pellet in Staining Buffer to count the cells using a hemocytometer.</li>
	<li>Resuspend the cells at a concentration of 5 &#215; 106 cells/mL and use them for surface staining (see section 2). 
	NOTE: For this purpose, plate the cells in a 96-well round-bottom plate followed by antibody staining and washes. If a plate centrifuge is not available, use flow tubes instead of plates. With this protocol, ~15-20 &#215; 106 cells per lung can be obtained from a 6-10-week-old C57BL/6 mouse of average size.</li>
</ol>
3. Surface antibody staining
<ol>
	<li>Transfer 1 &#215; 106 cells in 200 &#181;L per well in a 96-well plate. Centrifuge the plate at 350 &#215; g for 7 min at 4 &#176;C. In the meantime, prepare the Fc-block solution by diluting anti-16/32 antibody (1:100) in staining buffer (Table 1).</li>
	<li>Resuspend the cells in 50 &#181;L of the pre-prepared Fc-blocking solution (Table of Materials) and incubate for 15-20 min at 4 &#176;C or on ice.</li>
	<li>Add 150 &#181;L of staining buffer and centrifuge the plate at 350 &#215; g for 5 min at 4 &#176;C. Meanwhile, prepare the surface antibody cocktail by diluting surface antibodies (1:100; Table 2) in staining buffer. 
	NOTE: (i) Anti-16/32 antibody for Fc-blocking can be used with the surface antibodies in the same mixture. (ii) If fixable viability dye is used, add it to the surface antibody cocktail at a dilution of 1:1,000.</li>
	<li>Resuspend the cells in 50 &#181;L of the pre-prepared surface antibody cocktail and incubate for 30-40 min at 4 &#176;C in the dark. Wash the cells with staining buffer twice. 
	&#8203;NOTE: If no intracellular staining is required, resuspend the cells in 200 &#181;L of staining buffer and proceed directly to the acquisition of data on the flow cytometer. Alternatively, cells might be fixed and stored at 4 &#176;C for acquisition later. We recommend using the cells for flow cytometry within 24 h.</li>
</ol>
4. Cell fixation and intracellular staining
<ol>
	<li>Prepare the fixation/permeabilization buffer (Fix/Perm Buffer) by mixing three parts of fixation/permeabilization concentrate and 1 part of fixation/permeabilization diluent of the FoxP3/Transcription Factor Staining Buffer Set (Table 1).</li>
	<li>Resuspend the cells in 50 &#181;L of the pre-prepared Fix/Perm Buffer per well of the 96-well plate, where cells were plated as described in section 3, and incubate them for 20-25 min at 4 &#176;C in the dark.</li>
	<li>Dilute the 10x permeabilization buffer as 1: 10 in purified deionized water to prepare 1x permeabilization buffer.</li>
	<li>Wash the cells once with 1x permeabilization buffer. Meanwhile, prepare the intracellular antibody cocktail by diluting intracellular antibodies (1:100) in 1 mL of permeabilization buffer.</li>
	<li>Resuspend the cells using 50 &#181;L of the pre-prepared surface antibody cocktail per cell of the 96-well plate and incubate for 40 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells once with permeabilization buffer and once with staining buffer. After the final wash, resuspend the cells in 200 &#181;L of staining buffer. 
	NOTE: If no flow cytometer with plate reader is available, transfer the cells into flow cytometry tubes.</li>
	<li>Acquire a minimum of 1.5 &#215; 106 cells per sample on the flow cytometer. 
	NOTE: For single colors and unstained control samples, 0.5-1 &#215; 106 cells per sample will be sufficient. It is recommended to titer the individual antibodies used to achieve optimal staining and reduce costs. The present protocol has been optimized using Fix/Perm Buffer prepared using the FoxP3 staining buffer set. Because CD68 is a cytoplasmic and not a nuclear marker, other permeabilization solutions such as a low concentration of paraformaldehyde or cytofix/ cytoperm kits from various vendors might be sufficient.</li>
</ol>
Table 1: Buffers.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BSA buffer</td>
			<td>PBS + 0.5% Bovine serum albumin</td>
		</tr>
		<tr>
			<td>Digestion buffer</td>
			<td>Prewarmed (37 &#176;C) BSA buffer + 5 mg/mL collagenase type 1 + 0.2 mg/mL DNase I</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td>PBS + 2.5% FBS</td>
		</tr>
		<tr>
			<td>Fix/Perm Buffer</td>
			<td>Three parts of fixation/permeabilization diluent and 1 part of fixation/permeabilization diluent of the Foxp3/Transcription Factor Staining Buffer Set</td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td>10x permeabilization buffer from the Foxp3/Transcription Factor staining Buffer Set diluted 10 times in purified deionized water</td>
		</tr>
	</tbody>
</table>
Table 2: Usage of monoclonal antibodies.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Clone</td>
			<td>Fluorochrome</td>
			<td>Dilution</td>
			<td>Surface/intracellular</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>30-F11</td>
			<td>APC/CY7</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>Gr-1</td>
			<td>RB6-8C5</td>
			<td>BV421</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD68</td>
			<td>FA-11</td>
			<td>PerCPCy5.5</td>
			<td>1:100</td>
			<td>intracellular</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>M1/70</td>
			<td>PECy7</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>Siglec F</td>
			<td>S17007L</td>
			<td>FITC</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>N418</td>
			<td>BV650 or BV510</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD64</td>
			<td>X54-5/7.1</td>
			<td>PE/Dazzle 594</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>MHC-II</td>
			<td>M5/114.15.2</td>
			<td>AF700</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>2.00E+07</td>
			<td>PE / FITC</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Far Read Dead Cell Stain Kit</td>
			<td></td>
			<td>FarRed (APC) or Aqua (BV510)</td>
			<td>1:1000</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>17A2</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>B220</td>
			<td>RA3-6B2</td>
			<td>AF488</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>PK136</td>
			<td>FITC</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>30-F1</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>MERTK</td>
			<td>2B10C42</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>BM8</td>
			<td>BV605</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CX3CR1</td>
			<td>SA011F11</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>FcBlock (CD16/32)</td>
			<td>93</td>
			<td></td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL syringe plunger</td>
			<td>EXELINT</td>
			<td>26265</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needles</td>
			<td>BD Precision Glide Needle</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needles</td>
			<td>BD Precision Glide Needle</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Falcon</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>ThermoFisher</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Falcon/corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>ThermoFisher</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>Biolegend</td>
			<td>101215</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117339 / 117337</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103115</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD64</td>
			<td>Biolegend</td>
			<td>139319</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD68</td>
			<td>Biolegend</td>
			<td>137009</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse GR-1</td>
			<td>Biolegend</td>
			<td>108433</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse Siglec F</td>
			<td>Biolegend</td>
			<td>155503</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>AVERTIN</td>
			<td>Sigma-Aldrich</td>
			<td>240486</td>
			<td></td>
		</tr>
		<tr>
			<td>B220</td>
			<td>Biolegend</td>
			<td>103228</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>Biolegend</td>
			<td>121405 / 121419</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>Biolegend</td>
			<td>138503</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Biolegend</td>
			<td>100205</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>CX3CR1</td>
			<td>Biolegend</td>
			<td>149005</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123133</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>FcBlock (CD16/32)</td>
			<td>Biolegend</td>
			<td>101301</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Serrated Forceps</td>
			<td>Roboz Surgical Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>ThermoFisher</td>
			<td>00-5523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Futura Safety Scalpel</td>
			<td>Merit Medical Systems</td>
			<td>SMS210</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Far Read Dead Cell Stain Kit</td>
			<td>ThermoFisher</td>
			<td>L34973</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>MERTK</td>
			<td>Biolegend</td>
			<td>151505</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>MHC-II</td>
			<td>Biolegend</td>
			<td>107621</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>Biolegend</td>
			<td>108705</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>VWR</td>
			<td>Model 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td>SORVAL ST 16R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small curved scissor</td>
			<td>Roboz Surgical Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Christofides, A., et al. <a href="https://app.jove.com/v/62985">Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung.</a> J. Vis. Exp. (2021).
This video demonstrates an in vitro technique to identify immune cells residing in murine lungs. After isolating target immune cells from murine lung tissue, they are surface-labeled using a cocktail of fluorophore-tagged antibodies against specific markers on innate and adaptive immune cells. Additionally, monocytes, macrophages, and dendritic cells are labeled using an intracellular marker. The various immune cell types are then identified using flow cytometry.

<img alt="Figure 1" src="/files/ftp_upload/22141/22141_Figure1.jpg" /> 
Figure 1: The best cellular dissociation is achieved by 5 mg/mL of collagenase 1 in prewarmed PBS + 0.5% BSA. In all different conditions, 0.2 mg of DNAse I was also included. Abbreviations: BSA = bovine serum albumin; FSC-A = peak area of forward-scattered light; L/D = live/dead staining; SF = Siglec F.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take minced murine lung tissue in a digestion buffer and incubate at a physiological temperature.
Buffer enzymes break down the tissue matrix, loosening the cells.
Triturate to dissociate the cellular mass, and filter the suspension using a strainer to isolate the single cells.
Centrifuge to remove the supernatant with tissue debris. Incubate the cells with a lysis buffer to lyse the red blood cells.
Centrifuge to discard the cellular debris-containing supernatant. Resuspend the cells and transfer them to a microplate.
Add antibodies to block the Fc receptors, preventing background staining.
Introduce a fluorophore-labeled antibody cocktail targeting specific surface markers on the innate immune cells &#8212; monocytes, macrophages, dendritic cells, natural killer cells, neutrophils, eosinophils, and adaptive immune cells &#8212; B and T lymphocytes.
Fix and permeabilize the cells. Add fluorophore-labeled antibodies specific for an intracellular marker on monocytes, macrophages, dendritic cells, and eosinophils.
Using flow cytometry, classify the labeled immune cells.

21 Views. Un metodo in vitro per identificare le cellule immunitarie innate e adattative che risiedono nei polmoni murini

Video: Un metodo in vitro per identificare le cellule immunitarie innate e adattative che risiedono nei polmoni murini - Experiment

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

Citometria a flusso ad alta dimensionalità per l'analisi della funzione immunitaria di tessuti implantari sezionati

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

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JoVE Journal

Bioingegneria

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Immunologia e infezioni

Ibridazione in situ a fluorescenza per studiare la spermatogenesi delle zanzare

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Autore in primo piano: Ibridazione in situ a fluorescenza a montaggio intero per studiare la spermatogenesi nella zanzara Anopheles  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Ibridazione in situ a fluorescenza a montaggio intero per studiare la spermatogenesi nella zanzara Anopheles

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

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