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

Summary

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.

Abstract

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.

Introduction

Skin conditions ranging from contact dermatitis, eczema, psoriasis, cellulitis, fungal infections and abscesses to non-melanoma skin cancers were found to be among the 50 most prevalent diseases worldwide, and the fourth leading global cause of non-fatal diseases in 2010¹. Accordingly, the investigation of molecular and cellular mechanisms underlying diverse skin pathologies is a necessary and active area of research. Rodent models have been remarkably useful in the understanding of inflammatory skin conditions such as atopic dermatitis², psoriasis³, or Staphylococcus aureus infection⁴. Inexpensive, efficient, and simple protocols for the enzymatic digestion of mouse skin tissue can provide preparations of cells that can be used for a variety of downstream applications to better understand the pathophysiology of skin diseases. Here, a simple and economical method is described for enzymatic digest of mouse skin tissue and isolation of skin infiltrating leukocytes that can be used for cell culture, in vivo adoptive transfer, flow cytometric analysis and sorting or gene expression studies. The overall goal of this procedure is to prepare a single cell suspension of skin-infiltrating leukocytes with high cell viability while minimizing costs typically associated with custom reagent kits and mechanical dissociators.

Existing skin tissue dissociation methods^5-7 may result in low cell viability and surface marker integrity, or require custom enzyme kits and expensive tissue dissociation machines^8-11. While the digestion of mouse ear skin tissue is reasonably prevalent^12-13, digesting highly keratinized skin tissue (e.g. from the flank) can result in cell preparations contaminated with large amounts of non-cellular debris. In a recent study, Zaid and colleagues digested mouse flank skin for 90 min in 2.5 mg/ml dispase, followed by 45 min in 3 mg/ml collagenase⁷. In another study, these researchers used multiple incubations with a combined digestion of 2.5 hr, including the use of trypsin/EDTA, collagenase III, and dispase⁵. The use of trypsin is not recommended for enzymatic skin digestion, as treatment with trypsin from different manufacturers has been shown to measurably affect the integrity of cell surface proteins on mammalian cells^14-15. Additionally, dispase can have significant effects on proliferative abilities of CD4 and CD8α T cells and affect surface abundance of at least 20 molecules, including common T cell activation markers such as CD62L¹⁶. Other protocols use RPMI 1640 in the digestion medium⁶. However, the presence of Mg²⁺ and Ca²⁺ in RPMI can cause extensive cell aggregation¹⁷.

An ideal protocol for tissue dissociation should aim for high cell viability, low levels of cell aggregation, and minimal damage to cell surface proteins. High quality lymph node stromal cell preparations have been accomplished with protocols that use shorter enzyme incubations, Ca²⁺ and Mg²⁺ free media, and avoid trypsin and dispase¹⁸. However, protocols of this type have not been established for the dissociation of whole mouse skin.

Here, a protocol is described to dissociate, isolate, and enrich skin-infiltrating leukocytes from allergen-challenged mouse flank skin. Briefly, excised skin is pre-incubated in Hank's Balanced Salt Solution (HBSS) with 10% fetal bovine serum for 1 hr to soften the tissue for digestion and remove any excess dead skin or fatty tissue. This is followed by a 30 min enzymatic digestion step with 0.7 mg/mL collagenase D. Collagenase D has minimal effects on density of cell surface markers, and no effect on T cell proliferation in vitro^16,18, making it highly suitable for applications involving the characterization of surface proteins. Following enzymatic digestion, discontinuous density gradient centrifugation was used to remove epithelial cells and debris from the single-cell suspension and enrich for hematopoietic cells. Importantly, this procedure avoids expensive column-based magnetic cell separation reagents and tissue dissociation machines^8-11, and can be performed with equipment and materials found in a basic biomedical research laboratory. Here this protocol was used to isolate leukocytes from flank skin challenged three times with the hapten oxazolone (Ox) in previously sensitized ND4 Swiss mice (adapted from 19). Cells were analyzed using multi-parametric flow cytometry. This technique yielded a cell suspension with minimal debris and >95% viability of isolated lymphocytes which were analyzed by multi-parametric flow cytometry to measure the infiltration of T lymphocytes and neutrophils into the affected skin.

Protocol

Note: 8-12 week old female ND4 Swiss Webster mice, conventionally housed with free access to food and water, were used for these studies. The experimental protocol used (B13S1) was approved by Macalester College's IACUC.

1. Sensitization and Challenge with Oxazolone

1. Day 0
   1. Prepare anesthesia chamber by adding 3 ml isoflurane to absorbent towels placed beneath mesh at the bottom of a 4 L lidded glass jar. For the ND4 female mice used here, time in the chamber is 30-60 sec until the subject is adequately anesthetized; optimize anesthetic administration for the mouse strain being used.
   2. Shave each anesthetized mouse's back and flank using an animal hair trimmer.
2. Day 1
   1. Make a 2% solution of 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (Ox) into absolute ethanol, and incubate at 50 °C for 15 min on a rotating plate in an incubator.
   2. Briefly anesthetize mice using isoflurane anesthesia as described in 1.1.1 and apply 100 µl of 2% Ox to the shaved back in several serial applications of about 20 µl each. Wait 5-10 sec between serial applications for the solution to dry.
   3. Take care to avoid spilling the 2% Ox solution on regions other than the back, and wait for the solution to dry between applications.
3. Days 5-7
   1. Make a 1% solution of (Ox) in absolute ethanol, and incubate at 50 °C for 15 min on a rotating plate in an incubator.
   2. Gently but firmly immobilize the mouse at the scruff of the neck and tail base with abdomen facing up and neck slightly tilted downward (similar to a hold for an intraperitoneal injection) and apply 100 µl of 1% Ox to the shaved flank in several serial applications and taking the time to dry the skin afterwards as described above.
   3. For control mice, apply 100 µl of absolute ethanol vehicle to the shaved flank.

2. Flank Skin Harvest

1. Euthanize mice using carbon dioxide inhalation, and carefully excise the desired area of flank skin (~25 mm x 25 mm of skin) with 10 cm surgical scissors.
2. Using a clean, sharp surgical blade, scrape away the excess fat and connective tissue from the flank skin.
3. Place 3 pieces of flank skin from 3 mice into a 50 ml conical tube containing 10 ml RT HBSS [with phenol red, without calcium or magnesium, 5 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10% fetal bovine serum (FBS)].

3. Pre-digestion Tissue Washing

1. Place tube containing skin fragments on a rotating plate for 30 min in a dry non-gassed incubator maintained at 37°C.
2. Vortex vigorously for 10 sec at the end of the incubation period.
3. Strain the contents of the tube through a 70 µm strainer to discard the wash buffer and collect the tissue. A single strainer can be reused throughout the entire procedure within a biological treatment group.
4. Using a pair of forceps, transfer the flank skin from the strainer into a new 50 ml conical tube containing 10 ml of fresh, RT HBSS media (containing EDTA, HEPES, and FBS as described above).
5. With a 12.5 cm pair of surgical dissecting scissors immersed into the tube, mince each piece of flank skin so that the average piece of tissue is approximately 2.2 mm x 2.2 mm in area.
6. Place tube containing skin fragments on a rotating plate for 30 min in a dry non-gassed incubator maintained at 37°C.
7. Vortex vigorously for 10 sec at the end of the incubation

4. Collagenase Digestion of Skin

1. Strain the contents of the tube through a 70 µm strainer, collect and transfer the flank skin pieces into a new 50 ml conical tube containing 10 ml fresh, RT HBSS media supplemented with 0.7 mg/ml collagenase D.
2. Place tube containing digestion medium and skin fragments on a rotating plate for 30 min in a dry non-gassed incubator maintained at 37°C.
3. Vortex contents of tube vigorously for 10 sec at the end of the incubation.
4. Filter contents of tube through a 70 µm strainer into a new 50 ml conical tube; keep the flow-through containing the digested tissue and discard the strainer and flank skin debris.
5. Wash the flow-through in 50 ml of HBSS media, centrifuge for 5 min at 350 g, and decant the supernatant.

5. Density Gradient Centrifugation

1. Using a serological pipette, add 3 ml RT 67% density gradient centrifugation media in 1X phosphate buffered saline (PBS) into a new 15 ml conical tube for each sample.
2. Resuspend the cell pellet in 5 ml RT 44% density gradient centrifugation media in HBSS with phenol red.
3. Gently layer 5 ml of the 44% density gradient centrifugation media containing the cells on top of the 67% density gradient centrifugation media sample using a serological pipette. Make sure to set the pipettor to the slowest speed, and place the pipette along the wall of the conical tube. Be careful not to shake, disrupt, or invert the gradient.
4. Set acceleration to the lowest setting, disengage the brake, and centrifuge the gradient at 931 x g for 20 min at RT making sure that the centrifuge is balanced.
5. After centrifugation, carefully remove the gradient from the centrifuge, and gently transport to the lab bench holding the tube upright. Using a 5 ml plastic transfer pipette, remove the cell layer at the interface of the 44% and 67% density gradient centrifugation media and transfer to a new 15 ml conical tube. If the interface is not visible, simply remove about 2 ml of liquid at the 67%-44% boundary.
6. Fill the conical tube containing cells from the interface with 2% FBS in 1X ice-cold PBS, centrifuge cells for 5 min at 350 x g, and decant the supernatant.
   Note: This is the first time that the cells can be cooled/kept cold since digestion steps are at 37°C and density gradient centrifugation media steps are at RT. Cells can be resuspended in a 1:1 Trypan blue dilution and counts and viability information can be obtained at this point.

6. Blocking and Staining for Flow Cytometric Analysis

1. Block FcγRII/III receptors on cells to prevent non-specific antibody staining. For the experiments described here, make antibody master mixes in PBS with 2% FBS containing 1:100 dilution of unconjugated antibody against CD16/32 (2.4G2) to bind and block FcγRII/III receptors.
2. Incubate blocked cells with appropriate master mixes of antibodies against CD3ɛ (145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD44 (IM7), CD45 (30-F11), CD45R (RA3-6B2), CD62L (MEL-14), and Ly-6G/Ly-6C (Gr-1) (RB6-8C5), as well as Brilliant Violet 510 live/dead stain to identify dead cells. Use all antibodies either at the dilution recommended by manufacturers or previously optimized by researchers. For the experiments described here, use all antibodies at 1:100 dilution.
3. Wash cells and resuspend in 50 µl 1X PBS staining buffer with 2% FBS. Keep on ice until fluorescence data can be acquired on a flow cytometer.
   Note: It is important to have compensation parameters pre-optimized with a lymphoid tissue and a common cell surface antigen (like CD4 or CD8) before acquisition of fluorescence data.
4. To increase the number of cells for downstream flow cytometry analysis, acquire data from entire samples of stained cells.

7. Analyze Flow Cytometry Data using Standard Methods to Accomplish the Steps Outlined Below

1. First, gate on the lymphocyte region of the forward scatter (area) by side scatter (area) plot.
2. Then, select single cells using the forward scatter (width) by side scatter (width), to avoid analyzing doublets. This can also be accomplished with a forward scatter height by side scatter height plot.
3. Then, gate on lineage (CD11b, CD11c, and CD45R/B220)-negative and CD3-positive events, to exclude macrophages, dendritic cells, and B cells, respectively, and confine analyses to the T cell compartment, if the goal is, as it is here, to assess infiltrating T cells.
4. Next, gate on live cells that exclude the live/dead stain.
5. Finally, gate on the cell population of interest (see representative results section).

Results

Collagenase D treated splenocytes show similar levels of CD4 and CD8α on T cells when compared to media-treated controls
First, any potential effects of collagenase D on the frequency and surface abundance of lineage and activation markers on T cell subsets were assessed using secondary lymphoid tissue as a control. A suspension of splenocytes was obtained from ND4 mice and washed for 1 hr with HBSS media. Next, half the cells subject...

Discussion

Characterizing changes in skin-resident leukocytes in rodent models of skin diseases such as atopic dermatitis or psoriasis is important for understanding mechanistic connections between inflammatory cell influx and disease pathology. Here we describe an economical technique to isolate leukocytes from skin tissue with basic equipment found in most biomedical research labs. This relatively rapid technique avoids the use of expensive tissue dissociation machines and custom tubes and reagents, helping to conserve resources ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HBSS with phenol red, without calcium, without magnesium, liquid	Sigma Aldrich	55021C	Keep sterile until day of usage.
HEPES, ≥99.5% (titration)	Sigma Aldrich	H3375
EDTA disodium salt solution	Sigma Aldrich	E7889	Keep sterile until day of usage.
Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select	MidSci	S01520HI	Keep sterile until day of usage.
Percoll, pH 8.5-9.5 (25°C)	Sigma Aldrich	P1644	Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.
Trimmer Combo Kit	Kent Scientific	CL9990-1201	Use the larger trimmer for shaving the flank and back.
4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one	Sigma Aldrich	E0753-10G	Dissolve in 100% EtOH at 50°C for 15 minutes on a rotating plate.
Purified anti-mouse CD16/CD32 (2.4G2)	Tonbo Biosciences	70-0161	Antibody used to block non-specific antibody binding during antibody staining for flow cytometry
anti-CD3ε-BV650 (145-2C11)	Becton Dickinson	564378	Antibody used to stain cells for flow cytometry
anti-CD4-APC-eFluor780 (RM4-5)	eBioscience	47-0042-80	Antibody used to stain cells for flow cytometry
anti-CD8α-BV785 (53-6.7)	BioLegend	100749	Antibody used to stain cells for flow cytometry
anti-CD11b-eFluor450 (M1/70)	eBioscience	48-0112-80	Antibody used to stain cells for flow cytometry
anti-CD11c-eFluor450 (N418)	eBioscience	48-0114-80	Antibody used to stain cells for flow cytometry
anti-CD44-BV711 (IM7)	Becton Dickinson	563971	Antibody used to stain cells for flow cytometry
anti-CD45-FITC (30-F11)	Tonbo Biosciences	35-0451-U025	Antibody used to stain cells for flow cytometry
anti-CD45R-eFluor450 (RA3-6B2)	eBioscience	48-0452-80	Antibody used to stain cells for flow cytometry
anti-CD62L-PerCP-Cy5.5 (MEL-14)	BioLegend	104431	Antibody used to stain cells for flow cytometry
anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)	BioLegend	108407	Antibody used to stain cells for flow cytometry
Ghost Dye-BV510	Tonbo Biosciences	13-0870-T100	Viability dye for flow cytometry
LSR Fortessa	Becton Dickinson	N/A	Cell analyzer (18 parameters)
Collagenase D from Clostridium histolyticum	Roche Applied Science	11088858001	Aliquot lyophilized enzyme at 5 mg/ml in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.  Store immediately at -20°C for up to six months.
ND4 Swiss female mice	Harlan	0-32	8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College's IACUC guidelines.