This research was supported in part by the Intramural Research Program of the NIH, including the National Institute of Biomedical Imaging and Bioengineering. Disclaimer: The NIH, its officers, and employees do no recommend or endorse any company, product, or service.

NOTE: Figure 1 gives an overview of the flow cytometry protocol.
1) Reagent preparation
<ol>
	<li>Prepare media for diluting enzymes and for tissue culture.
	<ol>
		<li>Add 5 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution into 500 mL of RPMI medium and shake well. Store the medium at 4 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Calculate the volume of the enzyme solution. 
	&#8203;NOTE: The volume of the enzyme solu...

<ol>
	<li>Joung, Y. H. <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+implantable+medical+devices:+from+an+engineering+perspective.">Development of implantable medical devices: from an engineering perspective.</a> International Neurourology Journal. 17 (3), 98-106 (2013).</li><li>Langer, R., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymers+for+the+sustained+release+of+proteins+and+other+macromolecules.">Polymers for the sustained release of proteins and other macromolecules.</a> Nature. 263 (5580), 797-800 (1976).</li><li>Rolfe, B., et al., Eberli, D., 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=The+fibrotic+response+to+implanted+biomaterials:+implications+for+tissue+engineering.">The fibrotic response to implanted biomaterials: implications for tissue engineering.</a> Regenerative Medicine and Tissue Engineering-Cells and Biomaterials. , (2011).</li><li>Erdem, S., G&#252;r, M., Kaman, M. 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M., 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=Modulation+of+leukocyte+infiltration+and+phenotype+in+microporous+tissue+engineering+scaffolds+via+vector+induced+IL-10+expression.">Modulation of leukocyte infiltration and phenotype in microporous tissue engineering scaffolds via vector induced IL-10 expression.</a> Biomaterials. 35 (6), 2024-2031 (2014).</li><li>Graney, P. L., Lurier, E. B., Spiller, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+and+bioactive+factor+delivery+systems+for+the+control+of+macrophage+activation+in+regenerative+medicine.">Biomaterials and bioactive factor delivery systems for the control of macrophage activation in regenerative medicine.</a> ACS Biomaterials Science &amp; Engineering. 4 (4), 1137-1148 (2018).</li><li>Kontos, S., Grimm, A. J., Hubbell, 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=Engineering+antigen-specific+immunological+tolerance.">Engineering antigen-specific immunological tolerance.</a> Current Opinion Immunology. 35, 80-88 (2015).</li><li>Sadtler, K., Elisseeff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+the+scaffold+immune+microenvironment+using+flow+cytometry:+practices,+methods+and+considerations+for+immune+analysis+of+biomaterials.">Analyzing the scaffold immune microenvironment using flow cytometry: practices, methods and considerations for immune analysis of biomaterials.</a> Biomaterials Science. 7 (11), 4472-4481 (2019).</li><li>Baumgarth, N., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> Journal of Immunological Methods. 243 (1-2), 77-97 (2000).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical Flow Cytometry. , (2003).</li><li>Nolan, J. P., Condello, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+flow+cytometry.">Spectral flow cytometry.</a> Current Protocols in Cytometry. , (2013).</li><li>Wolf, M. 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=A+biologic+scaffold-associated+type+2+immune+microenvironment+inhibits+tumor+formation+and+synergizes+with+checkpoint+immunotherapy.">A biologic scaffold-associated type 2 immune microenvironment inhibits tumor formation and synergizes with checkpoint immunotherapy.</a> Science Translational Medicine. 11 (477), (2019).</li><li>Kahng, J., 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=Flow+cytometric+white+blood+cell+differential+using+CytoDiff+is+excellent+for+counting+blasts.">Flow cytometric white blood cell differential using CytoDiff is excellent for counting blasts.</a> Annals of laboratory medicine. 35 (1), 28-34 (2015).</li><li>Sionov, R. 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The authors have nothing to disclose.

This review describes a detailed methodology for isolating cells from biomaterial implants to obtain a uniform cell suspension. In addition, a detailed protocol has been provided for staining the cell suspension for multicolor flow cytometry, along with the steps for configuring a flow cytometer for optimal results. Cell isolation methods can involve multiple steps, often utilizing manual tissue dissection followed by enzymatic digestion with proteolytic enzymes to dissociate the extracellular matrix in the tissue and di...

Advances in the field of medicine have led to the frequent use of implanted materials for supporting the function or re-growth of damaged tissue1,2. These include devices such as pacemakers, reconstructive cosmetic implants, and orthopedic plates used for bone fracture fixation3,4. However, the materials used to make these implants and the locations in which they are implanted play important roles in determining the success of these implants5,6,7. As foreign bodies, these implants can generate an immune response from the host that can either lead to rejection or tolerance8. This factor has driven biomaterial research to generate materials that can attract the desired immune response after implantation9,10,11,12.
The immune response is an essential requirement in the field of regenerative medicine, where a tissue or an organ is grown around a biomaterial skeleton (scaffold) in a laboratory for the replacement of a damaged tissue or organ13,14,15,16. In regenerative medicine, the goal is to replace missing or damaged tissue through the use of cells, signals, and scaffolds, each of which can be greatly modulated by immune responses17. Furthermore, even when a lack of immune response is desired, it is very rarely an absence of immune activity rather than the presence of a regulatory profile that is desired18. Techniques such as flow cytometry can play a significant role in characterizing the pattern of immune response to various biomaterials used for coating implant devices or for developing scaffolds for tissue engineering19.
This information, in turn, will ultimately help in developing biomaterials for implants that can be well-tolerated by the immune system or in developing scaffolds that can play a constructive role in tissue engineering. Proper preparation of samples for analysis by flow cytometry is an important step for avoiding inaccurate results in immune characterization via fluorescence activated cell sorting20,21. Therefore, this review presents a detailed methodology that can be utilized for the isolation of cells from scaffold tissue, staining the cell suspension, and analysis by flow cytometry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Plate</td>
			<td>Fisher Scientific</td>
			<td>07-000-646</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Brilliant Stain Buffer Plus</td>
			<td>BD Biosciences</td>
			<td>566385</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix</td>
			<td>BD Biosciences</td>
			<td>554655</td>
			<td>For only fixing cells</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A7906</td>
			<td>For preparing FACS staining buffer</td>
		</tr>
		<tr>
			<td>CD11b AF700</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>Clone: M1/70</td>
		</tr>
		<tr>
			<td>CD11c PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>117325</td>
			<td>Clone: N418</td>
		</tr>
		<tr>
			<td>CD197 PE/Dazzle594</td>
			<td>Biolegend</td>
			<td>120121</td>
			<td>Clone: 4B12</td>
		</tr>
		<tr>
			<td>CD200R3 APC</td>
			<td>Biolegend</td>
			<td>142207</td>
			<td>Clone: Ba13</td>
		</tr>
		<tr>
			<td>CD206 PE</td>
			<td>Biolegend</td>
			<td>141705</td>
			<td>Clone: C068C2</td>
		</tr>
		<tr>
			<td>CD45 BUV737</td>
			<td>BD Biosciences</td>
			<td>612778</td>
			<td>Clone: 104/A20</td>
		</tr>
		<tr>
			<td>CD86 BUV395</td>
			<td>BD Biosciences</td>
			<td>564199</td>
			<td>Clone: GL1</td>
		</tr>
		<tr>
			<td>CD8a BV421</td>
			<td>Biolegend</td>
			<td>100737</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Comp Bead anti-mouse</td>
			<td>BD Biosciences</td>
			<td>552843</td>
			<td>For compensation control</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>11284932001</td>
			<td>Bovine pancreatic deoxyribonuclease I (DNase I)</td>
		</tr>
		<tr>
			<td>F4/80 PE/Cy7</td>
			<td>Biolegend</td>
			<td>123113</td>
			<td>Clone: BM8</td>
		</tr>
		<tr>
			<td>Fc Block</td>
			<td>Biolegend</td>
			<td>101301</td>
			<td>Clone: 93</td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Solution Kit</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>For fixing and permeabilization of cells.</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td>Buffer to supplement cell media</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Millipore Sigma</td>
			<td>5401127001</td>
			<td>Blend of purified Collagenase I and Collagenase II</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
			<td>Thermo Fisher</td>
			<td>L23105</td>
			<td>Viability dye</td>
		</tr>
		<tr>
			<td>Ly6c AF488</td>
			<td>Biolegend</td>
			<td>128015</td>
			<td>Clone: HK1.4</td>
		</tr>
		<tr>
			<td>Ly6g BV510</td>
			<td>Biolegend</td>
			<td>127633</td>
			<td>Clone: 1A8</td>
		</tr>
		<tr>
			<td>MHCII BV786</td>
			<td>BD Biosciences</td>
			<td>742894</td>
			<td>Clone: M5/114.15.2</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Thermo Fisher</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher</td>
			<td>11875176</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Siglec F BV605</td>
			<td>BD Biosciences</td>
			<td>740388</td>
			<td>Clone: E50-2440</td>
		</tr>
		<tr>
			<td>V-bottom 96-well plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The process of development of flow cytometry panels for immune analysis often relies on the comparison of results to existing data and the literature in the field. Knowledge of how populations may present in flow cytometry is critical for proper interpretation of data. Regardless, populations and cell types can appear differently in different tissues, so some variability is to be expected. In the context of well-defined control tissues, such staining optimization can be evaluated against ...

Watch this Scientific Journal Video about High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues at JoVE.com

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

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.

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

This protocol can help us characterizing host immune response against different biomaterials which can subsequently assist in designing better future medical implants. Flow cytometry gives us information about immune cells infiltrating a biomaterial which helps us determine mechanisms of how cells respond to injury and material implantation as well as targets for improved therapeutics. The same protocol can be modified to characterize immune response in different settings.Dissect the quad muscle of mice that received an ECM implant one week ago and collected in a 50 milliliter tube containing five milliliters of serum free media. Finally diced the tissue using scissors. Next, add five milliliters of digestive enzyme media to the tube.Place the tube containing digestive media and diced tissues in a shaking incubator for 45 minutes at 37 degrees Celsius and 100 RPM. At the end of the incubation filter the digested tissue suspension through a 70 micron strainer into an individual 50 milliliter tube. Use a five milliliter syringe head to mash any solid chunk of tissues and wash the strainer with room temperature PBS.Discard any remaining residue in the strainer and adjust the volume of the tube to 50 milliliters with room temperature PBS. Collect the cells by centrifugation and resuspend the pellets in 10 milliliters of cold five millimolar EDTA solution in PBS. If blood cells are present in the sample, resuspend the pellet in one milliliter of RBC lysis buffer, incubate for 10 minutes, then add nine milliliters of EDTA.Leave the suspension on ice for 10 minutes. Then adjust the volume to 50 milliliters with cold PBS. Collect the cells by centrifugation and resuspend the pellets and 100 microliters of cold PBS.Transfer 10 microliters of the suspension to individual micro centrifuge tubes and mix with 10 microliters of trip and blue for counting. Dispense the remaining volume of cells into each well of a V bottom 96 well plate. Remove 20 microliters of cells from the well and add them into a new well to use as unstained control.Then use PBS to bring the final volume in each well to 200 microliters. Collect the cells at the bottom of the plate wells by centrifugation and resuspend the cells in 100 microliters of a one to 1000 concentration of viability dye per well. At the end of the incubation, wash each well with 100 microliters of fresh PBS and resuspend the pellets in 200 microliters of staining buffer per well.After the second centrifugation, resuspend the cells in 50 microliters of one to 100 dilution of monocyte blocker. Incubate the cell suspension for five minutes on ice and then add 50 microliters of antibody cocktail. Incubate for 30 minutes on ice protected from light.Then wash the wells with 100 microliters of staining buffer per well. Centrifuge the cells again and wash with 200 microliters of staining buffer. Repeat the process two more times followed by resuspending the cells in 400 microliters of staining buffer.Before analyzing the sample, run an unstained sample to allow the cell population to be adjusted on a side scatter versus a forward scatter plot. Next, run the stained sample. Extract the autofluorescent signature.Then import the FCS files to use them as a control for unmixing. Click on the unmixing icon to open the unmixing wizard and perform unmixing using all single color controls by gating the positive and negative populations. Next, click on the QC section and look at the complexity index.The complexity index is a measure of how distinguishable a collection of spectral signatures is when the spectral signatures are unmixed together. Finally, unmixed the sample by clicking live unmix. Here, the results of a 14 color facts on control mouse tissue can be observed.In this analysis, several lobbyist populations, such as ly6g positive neutrophils and ly6c medium and high expressing monocyte classes could be observed. CD11c high MHC two positive dendritic cells were readily apparent when gated against CD11b to rule out macrophages and other myeloid lineage cells such as neutrophils and monocytes. A subset of CD206 positive CD86 positive dendritic cells could be identified by focusing on this CD11c positive population, which includes both CD86 high M1 like dendritic cells and CD206 high M2 like dendritic cell populations.F4/80 demonstrated a gradient of expression as is commonly observed in various macrophage populations. Siglec-F was present on both F4-80 positive and negative populations most likely corresponding to a macrophage subset and eosinophils respectively. Although staining with cell surface markers allows these designations of cell types to be made, it is important to note that cells expressing different markers should be viewed in a functional manner as opposed to a more binary classification.When attempting this protocol, keep in mind that understanding the autofluorescent signature of your understained samples before designing your panel is key in achieving a better and mixing. Besides analyzing cells, the isolated cells can be sorted into specific populations for downstream evaluations with cellular assays, in vitro, transcriptomic analysis, or for microscopic analysis to evaluate cellular morphology. Increasing complexity of flow cytometry analyses of biomaterials helps bridge the gap between basic immunology studies and the engineering of new therapeutics.

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2.0K vues.  National Institutes of Health. Ce protocole peut nous aider à caractériser la réponse immunitaire de l’hôte contre différents biomatériaux, ce qui peut par la suite aider à concevoir de meilleurs implants médicaux futurs. La cytométrie en flux nous donne des informations sur les cellules immunitaires qui infiltrent un biomatériau, ce qui nous aide à déterminer les mécanismes de réponse des cellules aux lésions et à l’implantation du matériau, ainsi que des cibles pour améliorer les traitements. Le mê...