Name: isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation
Uploaded: 2016-01-25T18:00:00
Description: Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

National Institutes of Health (NIH R15 NS067536-01A1 til DC), National Vulvodynia Association (pris til DC), og Macalester College støttet dette arbejde. CB modtog et stipendium fra Macalester College Beckman Scholars Program, finansieret af Arnold og Mabel Beckman Foundation. BTF og TM understøttes af JDRF 2-2011-662. Vi takker Dr. Jason Schenkel og Dr. Juliana Lewis for teknisk rådgivning, og alle nuværende og tidligere medlemmer af Chatterjea laboratorium for deres hjælp og støtte.

Bemærk: 8-12 uger gamle kvindelige ND4 Swiss Webster mus, konventionelt opstaldet med fri adgang til mad og vand, blev anvendt til disse undersøgelser Den eksperimentelle protokol, der bruges (B13S1) blev godkendt af Macalester akademiets IACUC.. 1. sensibilisering og udsættelse for oxazolon <ol><li> Dag 0 <ol><li> Forbered anæstesi kammer ved tilsætning af 3 ml isofluran absorberende håndklæder placeret under mesh nederst på en 4 L låg glaskrukke. For ND4 hunmus anvendes her, tid i kammeret ...

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Karakterisering forandringer i hud-resident leukocytter i gnavermodeller for hudsygdomme, såsom atopisk dermatitis eller psoriasis er vigtige for forståelsen af ​​mekanistiske forbindelser mellem inflammatorisk celleindstrømning og sygdom patologi. Her beskriver vi en økonomisk teknik til at isolere leukocytter fra hud væv med basisudstyr findes i de fleste biomedicinske forskningslaboratorier. Denne relativt hurtige teknik undgår brugen af ​​dyre væv dissociation maskiner og brugerdefinerede rør og reag...

Hudlidelser lige fra kontakteksem, eksem, psoriasis, cellulitis, svampeinfektioner og bylder til ikke-melanom hudkræft viste sig at være blandt de 50 mest udbredte sygdomme i hele verden, og den fjerde førende årsag til ikke-dødelige sygdomme i 2010 1. Derfor undersøgelse af molekylære og cellulære mekanismer bag diverse hud patologier er en nødvendig og aktivt forskningsområde. Gnavermodeller har været bemærkelsesværdigt nyttige i forståelsen af inflammatoriske hudtilstande, såsom atopisk dermatitis 2, psoriasis 3 eller Staphylococcus aureus infektion 4. Billige, effektive og simple protokoller for enzymatisk fordøjelse af muse hudvæv kan give præparater af celler, som kan anvendes til en række af downstream-applikationer for bedre at forstå patofysiologien af ​​hudsygdomme. Her er en enkel og økonomisk fremgangsmåde beskrevet for enzymatisk fordøjelse af musehudvæv og isolering af hud infiltrerende leukocytter, som kan anvendes til celledyrkning, in vivo adoptiv overførsel, flowcytometrisk analyse og sortering eller genekspressionsstudier. Det overordnede mål med denne procedure er at forberede en enkelt celle suspension af hud-infiltrerende leukocytter med høj cellelevedygtighed samtidig minimere omkostningerne typisk er forbundet med brugerdefinerede reagenskits og mekaniske dissociators. Eksisterende hud væv dissociation metoder 5-7 kan resultere i lav levedygtighed og overfladen cellemarkør integritet, eller kræve brugerdefinerede enzym kits og dyre væv dissociation maskiner 8-11. Mens fordøjelsen af museøre hudvæv er rimeligt udbredt 12-13, fordøje meget keratiniseret hudvæv (fx fra flanken) kan resultere i cellepræparationer forurenet med store mængder af ikke-celleaffald. I en nylig undersøgelse, Zaid og kolleger fordøjet mus flanke huden i 90 min i 2,5 mg / ml dispase, følgenwed med 45 minutter i 3 mg / ml collagenase 7. I en anden undersøgelse, disse forskere anvendes flere inkubationer med en samlet fordøjelse af 2,5 timer, herunder anvendelse af trypsin / EDTA, collagenase III og dispase 5. Anvendelsen af trypsin anbefales ikke til enzymatisk fordøjelse hud, som er blevet vist behandling med trypsin fra forskellige producenter til målbart påvirke integriteten af celleoverfladeproteiner på pattedyrceller 14-15. Derudover kan dispase få væsentlig indvirkning på proliferative evner CD4 og CD8a T-celler og påvirke overflade overflod af mindst 20 molekyler, herunder fælles T-celle aktivering markører såsom CD62L 16. Andre protokoller anvender RPMI 1640 i fordøjelsen medium 6. Tilstedeværelsen af Mg2 + og Ca2 + i RPMI, kan imidlertid forårsage omfattende celleaggregering 17. En ideel protokol for væv dissociation bør sigte efter høj cellelevedygtighed, lavniveauer af celleaggregering og minimal skade på celleoverfladeproteiner. Høj kvalitet lymfeknude stromale celle forberedelser er opnået med protokoller, der bruger kortere enzym inkubationer, Ca 2+ og Mg 2+ frie medier, og undgå trypsin og Dispase 18. Det er imidlertid ikke blevet etableret protokoller af denne type for dissociation af hele musehud. Her en protokol beskrevet til at dissociere, isolere, og berige hud-infiltrerende leukocytter fra allergen-udfordrede mus flanke hud. Kort fortalt, udskåret hud er præinkuberet i Hanks Balanced Salt Solution (HBSS) med 10% føtalt bovint serum i 1 time for at blødgøre væv til fordøjelsen og fjerne overskydende døde hud eller fedtvæv. Dette efterfølges af en 30 min enzymatisk fordøjelse takt med 0,7 mg / ml collagenase D. Collagenase D har minimale virkninger på tætheden af celleoverflademarkører, og ingen virkning på T-celleproliferation in vitro 16,18, hvilket gør detsærdeles velegnet til anvendelser, der involverer karakterisering af overfladeproteiner. Efter enzymatisk nedbrydning blev diskontinuerlig densitetsgradientcentrifugering anvendes til at fjerne epitelceller og rester fra encellede suspension og berige for hæmatopoietiske celler. Det er vigtigt, denne procedure undgår dyre kolonne-baserede magnetiske cellesepareringspræparater reagenser og væv dissociation maskiner 8-11, og kan udføres med udstyr og materialer, der findes i en grundlæggende biomedicinsk forskning laboratorium. Her blev denne protokol, der bruges til at isolere leukocytter fra flanken hud udfordret tre gange med haptenet oxazolon (Ox) i tidligere sensibiliserede ND4 Swiss mus (tilpasset fra 19). Celler blev analyseret ved anvendelse af multi-parametrisk flowcytometri. Denne teknik gav en cellesuspension med minimal snavs og&gt; 95% levedygtighed isolerede lymfocytter, som blev analyseret ved multi-parametrisk flowcytometri til måling af infiltration af T-lymfocytter og neutrofiler i de berørte ski.

<table border="0" cellpadding="0" cellspacing="0" width="1213">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">HBSS with phenol red, without calcium, without magnesium, liquid</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">55021C</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">HEPES, &#8805;99.5% (titration)</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">H3375</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">EDTA disodium salt solution</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">E7889</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select</td>
			<td style="width:179px;">MidSci</td>
			<td style="width:181px;">S01520HI</td>
			<td style="width:396px;">Keep sterile until day of usage.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">Percoll, pH 8.5-9.5 (25&#176;C)</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">P1644</td>
			<td style="width:396px;">Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:457px;">Trimmer Combo Kit</td>
			<td style="width:179px;">Kent Scientific</td>
			<td style="width:181px;">CL9990-1201&#160;</td>
			<td style="width:396px;">Use the larger trimmer for shaving the flank and back.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:181px;">E0753-10G</td>
			<td style="width:396px;">Dissolve in 100% EtOH at 50&#176;C for 15 minutes on a rotating plate.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:457px;">Purified anti-mouse CD16/CD32 (2.4G2)</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">70-0161</td>
			<td style="width:396px;">Antibody used to block non-specific antibody binding during antibody staining for flow cytometry</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:457px;">anti-CD3e-BV650 (145-2C11)</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">564378</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD4-APC-eFluor780 (RM4-5)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">47-0042-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD8a-BV785 (53-6.7)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">100749</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD11b-eFluor450 (M1/70)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0112-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD11c-eFluor450 (N418)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0114-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD44-BV711 (IM7)</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">563971</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD45-FITC (30-F11)</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">35-0451-U025</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD45R-eFluor450 (RA3-6B2)</td>
			<td style="width:179px;">eBioscience</td>
			<td style="width:181px;">48-0452-80</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-CD62L-PerCP-Cy5.5 (MEL-14)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">104431</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)</td>
			<td style="width:179px;">BioLegend</td>
			<td style="width:181px;">108407</td>
			<td style="width:396px;">Antibody used to stain cells for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">Ghost Dye-BV510</td>
			<td style="width:179px;">Tonbo Biosciences</td>
			<td style="width:181px;">13-0870-T100</td>
			<td style="width:396px;">Viability dye for flow cytometry</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:457px;">LSR Fortessa</td>
			<td style="width:179px;">Becton Dickinson</td>
			<td style="width:181px;">N/A</td>
			<td style="width:396px;">Cell analyzer (18 parameters)</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:457px;">Collagenase D from Clostridium histolyticum</td>
			<td style="width:179px;">Roche Applied Science</td>
			<td style="width:181px;">11088858001</td>
			<td style="width:396px;">Aliquot lyophilized enzyme at 5 mg/mL in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.&#160; Store immediately at -20&#176;C for up to six months.</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:457px;">ND4 Swiss female mice</td>
			<td style="width:179px;">Harlan</td>
			<td style="width:181px;">0-32</td>
			<td style="width:396px;">8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College&#39;s IACUC guidelines.&#160;</td>
		</tr>
	</tbody>
</table>

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.

Collagenase D behandlede splenocytter viser lignende niveauer af CD4 og CD8 α på T-celler i forhold til medierne-behandlede kontroller Først blev den eventuelle påvirkning af collagenase D om hyppigheden og overflade overflod af afstamning og aktivering markører på T-celle delmængder vurderet ved hjælp sekundær lymfoide væv som kontrol. En suspension af splenocytter blev opnået fra ND4 mus og vasket i 1 time med HBSS me...

Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

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

Isolering af leukocytter fra musehud anvendelse af enzymatiske Digest og Gradient Separation

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 ...

The overall goal of this procedure is to isolate skin-infiltrating leukocytes using enzymatic digestion and density gradient separation. This method can be applied to the investigation of cellular and molecular mechanisms in many mouse models of skin pathologies, including atopic dermatitis. Though this method can provide insight into skin-infiltrating leukocyte biology, it can also be applied to the investigation of other resident cell populations.Generally, individuals new to this method will struggle with the skin harvesting, which includes a complete removal of the excess adipose and connective tissues. Visual demonstration of this method is critical, as skin harvesting and density gradient handling are difficult to learn, and require precision and practice. At the appropriate time point after treatment, use 10 centimeter surgical scissors to carefully excise a 25 by 25 millimeter area of shaved flank skin from an adult eight to twelve-week-old female ND4 Swiss-Webster mouse.Use a clean, sharp surgical blade to scrape away the excess fat and connective tissue from the skin, then transfer the skin into a 50 milliliter conical tube containing 10 milliliters of room temperature HBSS supplemented with EDTA, HEPES, and FBS, and predigest the tissue on a rotating plate for 30 minutes in a dry, non-gassed, 37 degree Celsius incubator. At the end of the incubation, vortex the tube vigorously for ten seconds and filter the tissue slurry through a 70 micron strainer. Next, use a pair of forceps to transfer the flank skin pieces from the strainer into a new 50 milliliter tube containing ten milliliters of fresh, room temperature HBSS media, supplemented with EDTA, HEPES, and FBS, then use a 12.5 centimeter pair of surgical dissecting scissors to mince each piece of flank skin so that the average piece of tissue is approximately 2.2 by 2.2 millimeters in size.When all of the pieces have been minced, return the tube to the rotating plate for another 30 minute incubation. Vortex the tube vigorously for 10 seconds at the end of the incubation and filter the contents through the 70 micron strainer. Collect the remaining flank skin pieces in a new 50 milliliter tube containing 10 milliliters of HBSS supplemented with 0.7 milligrams per milliliter of Collagenase D, and return the tube to the rotating plate for another 30 minute incubation.After another 10 seconds of vortexing, filter the contents of the tube again, this time discarding the debris on the strainer, then fill the tube with up to 50 milliliters of HBSS, EDTA, HEPES, FBS, spin down the cells and decant the supernatant. Add three milliliters of room temperature, 67 percent density gradient centrifugation medium in PBS to a new 15 milliliter conical tube, and resuspend the cell pellet in five milliliters of room temperature 44 percent density gradient centrifugation medium in HBSS with phenol red. Setting the pipettor to the lowest speed, place the pipette on the side of the tube and gently layer the cells on top of the 67 percent density gradient centrifugation medium.When working with density gradients, take care to avoid making bubbles and to minimize any disruptions to the gradient interface. Then separate the cells, using a five milliliter transfer pipette to collect the cells at the interface at the end of the centrifugation. As gently layering the gradient and carefully extracting the interface of the gradients are critical steps, it is wise to practice the density gradient centrifugation medium layering and interface removal with the suspension of murine splenocytes before attempting the procedure with the experimental cells of interest.Finally, wash the collected cells in ice-cold PBS supplemented with FBS in a 15 milliliter conical tube and count the number of viable cells by trypan blue exclusion. CD4 and CD8-alpha surface expression are not affected by Collagenase D digestion, as the same number of CD4 positive and CD8-alpha positive cells is detected with or without enzymatic digestion. Further, Collagenase D digestion exhibits no effect on the surface density of the T cell activation markers CD44 and CD62L on CD4 positive T cells.After gradient separation, about 250 viable immune cells per square millimeter of flank tissue are isolated from Oxazolone, or Ox-challenged mice, versus only four viable immune cells per square millimeter of tissue in vehicle-challenged mice, previously sensitized with Ox.95 percent of the low forward and side scatter single cells in Ox-challenged mice, and 71 percent of these cells in vehicle-challenged controls are live CD45 positive cells, equivalent to an approximately 67 fold increase in the infiltrating CD45 positive immune cell population in the flank skin following three daily Ox challenges. Indeed, Ox challenge results in the pronounced infiltration of T cells and neutrophils into the flank skin, with 42 percent of the single, live CD45 positive cells in Ox-treated animals, apparent Gr-1 positive neutrophils, and approximately 84 percent CD3 positive T cells expressing CD8-alpha, or CD4, compared to control, ethanol-treated animals. Once mastered, this technique can be completed in about three hours if it's performed properly.While attempting this procedure, it's important to remember to handle the density gradient carefully. Don't forget to always use room temperature density gradient centrifugation medium when performing this procedure. Following this procedure, other methods like flow cytometric analysis, cell sorting, cell transfer, or in vitro cell culture can be performed to evaluate the phenotype and function of the skin-infiltrating leukocytes.After watching this video, you should have a good understanding of how to isolate skin-infiltrating leukocytes using enzymatic digestion and density gradient centrifugation.

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