We would like to thank Dr. Fadia Kamal, and Dr. Reyad Elbarbary for allowing us to use lab instruments and technical support. This work was supported by grants from the NIH (K08 AR060164-01A) and DOD (W81XWH-16-1-0725) to J. C. E. in addition to institutional support from the Pennsylvania State University Hershey Medical Center.

Acquisition and use of de-identified human foreskin tissue for research purposes was reviewed and received the determination of &#34;not human research&#34; by the Penn State College of Medicine Institutional Review Board (IRB #17574).
NOTE: By following the protocol below, 2.4 x 106 keratinocytes, 4.4 x 106 fibroblasts and 1.1 x 106 SCs are obtained from a single foreskin. In general, these primary cells can be used for 3 passages, depending on the experimental conditions.
1. Isolation of primary cells and culture conditions
<ol>
	<li>Epidermis and dermis separation procedure
	<ol>
		<li>Acquire neonatal foreskin following standard of care procedures by a physician under approved hospital regulatory protocols. The physician determines the timing of circumcision after birth and the amount of skin removed.</li>
		<li>Place the excised foreskin in a microcentrifuge tube containing 8-10 mL of ice-cold Dulbecco&#39;s Modified Eagle Medium (DMEM) basal medium and immediately transport the foreskin to the cell culture laboratory for cells isolation. Rinse the skin twice with 20-25 mL of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) containing 1x antibiotic/antimycotic. 
		NOTE: For maximum cell viability, the excised foreskin should be placed at 4 &#176;C until processing, and cells should be harvested within 24 h.</li>
		<li>After rinsing in DPBS/antibiotics, soak the foreskin in 25-30 mL of ice-cold DMEM basal medium for 10-15 min in a 10 cm tissue culture dish. Perform all processing steps in a tissue culture hood using sterile technique.</li>
		<li>Using a scalpel, carefully slice the foreskin open and expose the dermis and subcutaneous adipose tissue.</li>
		<li>Remove the subcutaneous adipose tissue using forceps, scalpel blade, and scissors, as needed. Discard the adipose tissue. Cut the cleaned foreskin into three to five smaller pieces.</li>
		<li>Transfer skin pieces to a sterile conical tube containing 16-20 mL of dispase-I/DMEM medium (4 mg/mL in DMEM basal medium).</li>
		<li>Mix the skin pieces in the conical tube by inversion, intermittently, during the first 30 min of incubation with dispase-I. Then place the conical tube at 4 &#176;C for 16-18 h, being mindful of immersing the skin pieces in dispase-I/media. 
		NOTE: Dispase-I solution should be prepared just before addition using cold DMEM basal medium, filtered with a 0.22 &#181;m strainer, and kept at 4 &#176;C.</li>
		<li>After overnight digestion with dispase-I, remove the skin pieces and place them on a dry, sterile, culture dish. Unroll each piece of skin so that the outer epidermal layer is touching the culture dish.</li>
		<li>Using two sets of forceps, carefully separate the epidermis from the dermis. Start on the edge of the tissue and peel the epidermis away from the dermis. 
		NOTE: If the epidermis layer is difficult to separate from the dermis, the dispase-I digestion was not sufficient. Troubleshooting options: 1) return skin pieces to dispase-I/DMEM solution and incubate for an additional 2-3 h at 4 &#176;C; 2) place skin pieces in fresh dispase-I/DMEM and incubate at 4 &#176;C for 2-3 h more.</li>
		<li>Use the separated epidermis and dermis to isolate keratinocytes (epidermis) and fibroblasts and Schwann cells (dermis). 
		NOTE: Follow the steps below for specific cell types isolation and culture conditions.</li>
	</ol>
	</li>
	<li>Keratinocyte isolation from the epidermis
	<ol>
		<li>Add 5 mL of HEPES buffer saline (HBS) to a 10 cm culture dish and add the separated epidermis fragments. Incubate at room temperature (RT) for 10 min.</li>
		<li>After HBS incubation, collect the HBS solution in a 50 mL conical tube. Add 5 mL of trypsin neutralization buffer (TNB) (Table of Materials) in a 50 mL conical tube and set aside.</li>
		<li>Add 3 mL of trypsin solution to the epidermal fragments. Incubate at 37 &#176;C for 10 min.</li>
		<li>Gently agitate the epidermal fragments using forceps until the trypsin solution becomes turbid.</li>
		<li>Add the trypsin solution/keratinocytes to the HBS and TNB containing tube in step 1.2.2.</li>
		<li>For any remaining epidermal fragments that have not dissolved in the initial trypsin digestion, add 3 mL of 0.25% trypsin/Ethylenediaminetetraacetic acid (EDTA). Incubate at 37 &#176;C for 10 min and repeat steps 1.2.4 and 1.2.5.</li>
		<li>Add 2 mL of fetal bovine serum (FBS) to the trypsin/EDTA solution containing HBS and TNB solution tubes.</li>
		<li>Centrifuge the collection tube containing HBS/TNB/trypsin and keratinocytes at 870 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the cell pellet in 3 mL of keratinocyte complete growth medium (Table of Materials).</li>
		<li>Filter the cell suspension with a sterile 100 &#181;M filter into a sterile 50 mL conical tube to remove debris.</li>
		<li>Quantify the cell number and cell viability. 
		NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
		<li>Seed culture plates as needed for experiments. The recommended seeding density is ~45,000 cells/cm2.</li>
		<li>Place the culture plates in an incubator at 37 &#176;C in 5% CO2 for 2 days to allow keratinocytes to adhere to the dish.</li>
		<li>After 2 days, aspirate any non-adhered cells. Refresh the cell culture media every other day until keratinocytes reach desired confluence for passaging or downstream experiments (50%-80% confluence).</li>
	</ol>
	</li>
	<li>Isolation of Schwann cells (SCs) and fibroblasts from the dermis
	<ol>
		<li>Pre-coat T25 flasks with poly-L-lysine (PLL, 0.01 &#181;g/&#181;L) using 5 mL of 1x DPBS. Place the pre-coated flasks at 37 &#176;C for 3 h. Wash the PLL coating matrix twice using 1x DPBS (5 mL). 
		NOTE: T25 flasks can be prepared ahead of time.</li>
		<li>Rinse the isolated pieces of the dermis with 5 mL of DMEM basal medium.</li>
		<li>Mince the dermis into small pieces with scissors or a scalpel.</li>
		<li>Digest the minced dermis with 5 mL of collagenase (2 mg/mL in DMEM basal medium) at 37 &#176;C for 2.5 h. Gently triturate the minced dermis with a pipette tip every 30 min until the dermis completely dissociates.</li>
		<li>Filter the cell suspension with a 70 &#181;M cell strainer. It is necessary to apply mechanical force using a 1 mL syringe to fully filter the suspension.</li>
		<li>Dilute the filtered cell suspension with 5 mL of complete DMEM medium (DMEM basal medium + 5% FBS + 1x of antibiotic) to stop the enzymatic activity of collagenase.</li>
		<li>Centrifuge the cell suspension at 870 x g for 5 min at RT to pellet the cells.</li>
		<li>From this cell pellet, both fibroblasts and SCs will be obtained. Resuspend the cell pellet in 2 mL of DMEM complete medium to divide the cells (1 mL cell suspension/tube) and centrifuge at 870 x g for 5 min at RT.</li>
		<li>For SCs culture, follow steps 1.3.10-1.3.17 and for fibroblast culture, follow steps 1.3.18-1.3.22.</li>
		<li>From step 1.3.8, aspirate the supernatant. Resuspend the cell pellet in 5 mL of fresh DMEM complete medium.</li>
		<li>Quantify the cell number and viability. 
		NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
		<li>Seed the pre-coated T25 flasks with 5 mL of resuspended cells at a density 4.0 x 103 cells/mL. Incubate the flask at 37 &#176;C in the presence of 5% CO2 overnight (~12-16 h incubation).</li>
		<li>After 16 h, confirm cell adhesion by microscopy (Table of Materials) at 10x magnification.</li>
		<li>Remove the non-adherent cells and wash the flask 3x with 5 mL of 1x DPBS.</li>
		<li>Add 5 mL of 10 &#181;M cytosine arabinoside (antimitotic agent, kills fibroblasts) in DMEM complete medium. Incubate the flask at 37 &#176;C in the presence of 5% CO2&#160;for 24 h.</li>
		<li>Aspirate the cytosine arabinoside containing medium from the flask. Rinse the flask 3x with 5 mL of 1x DPBS.</li>
		<li>Refill the culture flask with 5 mL of SCs complete culture medium (Table of Materials) and incubate at 37 &#176;C in the presence of 5% CO2&#160;for 48 h. Refresh SC complete culture medium every other day until SCs reach 80% confluence.</li>
		<li>Aspirate the supernatant from the dissociated dermal layer (step 1.3.8) and resuspend the cell pellet with 5 mL of fibroblast complete medium (fibroblast basal medium + 5% FBS + 1x antibiotic).</li>
		<li>Quantify cell number and cell viability. 
		&#8203;NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
		<li>Plate the fibroblasts at a cell density of 4.0 x 103 cells/mL in T25 culture flasks (not pre-coated) using 5 mL of fibroblast complete medium. Incubate at 37 &#176;C in the presence of 5% CO2 overnight (~12-16 h incubation).</li>
		<li>After 16-24 h, confirm cell adhesion by microscopy (Table of Materials) at 10x magnification. Aspirate non-adherent cells from the flask and wash the flask twice with 5 mL of 1x DPBS.</li>
		<li>Add 5 mL of fresh fibroblast complete medium and incubate at 37 &#176;C in the presence of 5% CO2 for 48 h. Refresh the culture medium every other day until fibroblasts reach the desired confluence for passaging or downstream assays (50%-80% confluence).</li>
	</ol>
	</li>
</ol>
2. Verification of epidermal keratinocytes, dermal SCs and fibroblasts protein marker by immunofluorescence
<ol>
	<li>Day 1- Coat chamber glass slides and detach cells
	<ol>
		<li>Pre-coat 4-well chamber glass slides with collagen-I for keratinocytes, or PLL for SCs or, only 1x DPBS for fibroblasts (0.5 mL each well). Ensure to coat the entire surface. Incubate chamber slides at 37 &#176;C for 2 h. During incubation, prepare the cells.</li>
		<li>After cultured cells reach 50%-80% confluence, wash the flasks with 1x DPBS 3x (1 mL for each well).</li>
		<li>Detach the adhered cells with 5 mL of 0.25% trypsin-EDTA (TE) solution by incubating at 37 &#176;C in the presence of 5% CO2 for 2 min, until cells start to become round. Visualize cell detachment with microscopy (Table of Materials) at 10x magnification.</li>
		<li>After incubation, transfer TE solution containing cells to a conical tube with 5 mL fetal bovine serum (FBS).</li>
		<li>Add 5 mL of trypsin neutralization buffer (TNB) to the culture flask and then transfer the contents to the above-mentioned centrifuge tube (cells with TE and FBS). Visualize cell detachment with microscopy (Table of Materials) at 10x magnification. 
		NOTE: TNB or FBS or both can be used to neutralize trypsin activity.</li>
		<li>Centrifuge the conical tube containing cells/TE/TNB at 870 x g for 5 min. Transfer the supernatant to another tube and resuspend the cell pellet in the corresponding complete culture medium. Measure the cell viability as mentioned in step 1.2.11.</li>
		<li>Rinse the pre-coated chamber glass slides (step 2.1.1) with 1x DPBS (1 mL for each well) 3x.</li>
		<li>Semi-dry the slides inside the tissue culture hood and seed keratinocytes, SCs, or fibroblasts at an appropriate density (30,000 cells/0.5 mL/chamber) in an appropriate complete cell culture medium.</li>
		<li>Incubate the chamber slides at 37 &#176;C overnight to allow cells to adhere.</li>
	</ol>
	</li>
	<li>Day 2- Blocking the chamber glass slide with Bovine Serum Albumin (BSA) and primary antibodies incubation
	<ol>
		<li>Aspirate the cell media from the chamber slides and wash 3x with 1 mL of 1x DPBS.</li>
		<li>Fix the adhered cells with 4% paraformaldehyde in 1x DPBS (1 mL for each well) and incubate for 15 min at RT inside the tissue culture hood.</li>
		<li>Aspirate 4% paraformaldehyde from the chamber slides and wash the chamber slides 3x with 1x DPBS (1 mL for each well).</li>
		<li>Permeabilize the cell membranes with 1% TritonX-100 in 1x DPBS (1 mL for each well) and incubate for 15 min at RT.</li>
		<li>Aspirate the blocking solution from the chamber slides, wash 3x with 1x DPBS (1 mL for each well) for 30 s and then aspirate 1x DPBS from the well.</li>
		<li>Block the chamber slides with 5% BSA (0.5 mL for each well) and incubate for 45 min at RT.</li>
		<li>Aspirate the blocking solution from the chamber slides, wash 3x with 1x DPBS (1 mL for each well) for 30 s each and then aspirate 1x DPBS from the well.</li>
		<li>Dilute the primary antibodies with 5% BSA (0.2 mL for each well) (Primary antibodies dilutions: Keratinocytes: mouse cytokeratin 14 antibody (1:100) and, cytokeratin 10 antibody (1:100); Schwann cells: mouse S100 antibody (1:200), and rabbit nerve growth factor receptor (p75-NTR) antibody (1:500); Fibroblasts: rabbit vimentin antibody (1:200), and mouse alpha-smooth muscle actin (&#945;-SMA) antibody (1:200)).</li>
		<li>Add the primary antibodies to the corresponding cells on the chamber glass slide and incubate for about 15-16 h (overnight) at 4 &#176;C in a humidified chamber.</li>
		<li>Aspirate the primary antibodies from the chamber slides, wash 3 times with 1x DPBS (1 mL for each well) for 30 s each and then aspirate 1x DPBS from the well.</li>
		<li>Dilute the secondary antibodies (Goat anti-rabbit IgG Secondary Antibody-Alexa Fluor 488 (1:500) and Goat anti-Mouse IgG Secondary Antibody-Alexa Fluor 594 (1:500)) with 0.5% BSA. Add appropriate secondary antibody to each well (0.2 mL for each well). Incubate for 45 min at RT.</li>
		<li>Aspirate the secondary antibody from chamber slides, wash 3x with 1x DPBS (1 mL for each well) for 30 s each and then aspirate 1x DBPS from the well.</li>
		<li>Remove the gasket on the 4-well chamber slide using gasket remover. Try not to leave any adhesive on the slide as this interferes with coverslip application. Add a drop of mounting medium with 4&#39;,6-diamidino-2-phenylindole (DAPI). Alternatively, counter-stain the wells with DAPI and use a non-label-containing mounting medium.</li>
		<li>Carefully place the coverslip on the glass slide by avoiding air bubbles and seal the end of the coverslip with glue. Observe immunofluorescence staining patterns by microscopy (Table of Materials) at 10x and/or 20x magnification.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+disadvantages+of+using+human+cells+for+pharmacological+and+toxicological+studies.">Advantages and disadvantages of using human cells for pharmacological and toxicological studies.</a> Human &amp; Experimental Toxicology. 13 (8), 568-573 (1994).</li><li>Green, H., Kehinde, O., Thomas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+cultured+human+epidermal+cells+into+multiple+epithelia+suitable+for+grafting.">Growth of cultured human epidermal cells into multiple epithelia suitable for grafting.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (11), 5665-5668 (1979).</li><li>Fuchs, E., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+keratin+gene+expression+during+terminal+differentiation+of+the+keratinocyte.">Changes in keratin gene expression during terminal differentiation of the keratinocyte.</a> Cell. 19 (4), 1033-1042 (1980).</li><li>Aasen, T., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+human+keratinocytes+from+skin+or+plucked+hair+for+the+generation+of+induced+pluripotent+stem+cells.">Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells.</a> Nature Protocols. 5 (2), 371-382 (2010).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> Journal of Visualized Experiments: JoVE. (44), e2033 (2010).</li><li>Belviso, I., 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=Isolation+of+adult+human+dermal+fibroblasts+from+abdominal+skin+and+generation+of+induced+pluripotent+stem+cells+using+a+non-integrating+method.">Isolation of adult human dermal fibroblasts from abdominal skin and generation of induced pluripotent stem cells using a non-integrating method.</a> Journal of Visualized Experiments: JoVE. (155), e60629 (2020).</li><li>Silva, W. N., 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=Role+of+Schwann+cells+in+cutaneous+wound+healing.">Role of Schwann cells in cutaneous wound healing.</a> Wound Repair and Regeneration. 26 (5), 392-397 (2018).</li><li>Bray, E. R., Cheret, J., Yosipovitch, G., Paus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+as+underestimated,+major+players+in+human+skin+physiology+and+pathology.">Schwann cells as underestimated, major players in human skin physiology and pathology.</a> Experimental Dermatology. 29 (1), 93-101 (2020).</li><li>Laverdet, B., 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=Skin+innervation:+important+roles+during+normal+and+pathological+cutaneous+repair.">Skin innervation: important roles during normal and pathological cutaneous repair.</a> Histology and Histopathology. 30 (8), 875-892 (2015).</li><li>Jessen, K. R., Mirsky, R., Lloyd, A. 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L., 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=Signals+for+proinflammatory+cytokine+secretion+by+human+Schwann+cells.">Signals for proinflammatory cytokine secretion by human Schwann cells.</a> Journal of Neuroimmunology. 101 (1), 47-60 (1999).</li><li>Kumar, A., Brockes, J. 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All other authors declare that they have no competing financial interests.

This protocol describes a method to isolate three distinct cell populations from a single piece of the foreskin, namely keratinocytes, fibroblasts, and Schwann cells. There are a few isolation protocols available to isolate keratinocytes and fibroblasts2,3,5,6, but none describe SC isolation. Apart from the key structural cells in skin, keratinocytes, and fibroblasts, skin is also highly innerv...

Primary cells derived from living tissue and cultured under in vitro conditions closely resemble the physiological state1, making them an ideal model for investigating physiological and pathophysiological processes. The skin contains multiple cell types, including keratinocytes, fibroblasts, sebocytes, melanocytes, and Schwann cells (SCs), which can be isolated and cultured for in vitro experiments. Methods to isolate and culture keratinocytes, fibroblasts, and SCs, from a single piece of skin, have not been described. The goal of this protocol is twofold: 1) to establish a reliable and reproducible method for the isolation of and culturing of dermal SCs and 2) to use an efficient, robust method for the isolation of keratinocytes, fibroblasts, and SCs from a single human foreskin.
At present, there are established protocols to isolate skin keratinocytes2,3,4 and fibroblasts5,6. These studies describe the isolation of either keratinocytes, fibroblasts, or both from the skin, but no protocol addresses how to establish cultures of primary SCs from human skin. Recent studies suggest that neuronal SCs modulate keratinocyte and fibroblast cellular processes and regulate normal skin physiological functions7. SCs are thus critical to skin homeostasis and contribute substantially to the regulate physiology that influences the behavior of neighboring skin cell types present8. Therefore, a protocol that allows for the isolation of each of these cell types is ideal for in vitro experiments involving cell-cell communication or cross-talk between cell types.
This protocol describes the establishment of individual cell cultures of primary cells from a single piece of skin. This protocol is particularly useful when the amount of tissue available is limited. Moreover, isolation of all three cell types from a single donor allows for robust comparisons between cell types or co-culture experiments while mitigating the influence of genetics during the desired experiment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;M sterile filters (Millex-GP Syringe Filter Unit,polyethersulfone)</td>
			<td>MilliporeSigma</td>
			<td>SLGPR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;M cell strainers</td>
			<td>CELLTREAT</td>
			<td>229483</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;M cell strainers</td>
			<td>CELLTREAT</td>
			<td>229485</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL disposable syringes</td>
			<td>BD Luer-Lok</td>
			<td>BD-309659</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL disposable syringes (Syringe sterile, single use)</td>
			<td>BD Luer-Lok</td>
			<td>BD309646</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL disposable syringes</td>
			<td>BD Luer-Lok</td>
			<td>BD305462</td>
			<td></td>
		</tr>
		<tr>
			<td>1% TritonX-100</td>
			<td>Sigma</td>
			<td>X100-1L</td>
			<td>Prepared at the time of use</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution</td>
			<td>ThermoFisher Scientific</td>
			<td>J19943.K2</td>
			<td>Ready to use and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>5% BSA</td>
			<td>Sigma</td>
			<td>A7906-100G</td>
			<td>Prepared at the time of use</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic</td>
			<td>ScienCell Research</td>
			<td>503</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemometec Vial1-Cassette</td>
			<td>Fisher Scientific</td>
			<td>NC1420193</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Gibco</td>
			<td>17018-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Fisherbrand</td>
			<td>12544D 22*50-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Sigma-Aldrich</td>
			<td>D46693</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basal medium</td>
			<td>ScienCell Research</td>
			<td>9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline free from Ca2+ and Mg2+ (DPBS)</td>
			<td>Corning</td>
			<td>&#160;21-031-CV)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>02-681-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast complete medium</td>
			<td>ScienCell Research</td>
			<td>2331</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A11032</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr>
			<td>Hanks&#39; Buffered Saline Solution (HBSS buffer)</td>
			<td>Lonza</td>
			<td>CC-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Human foreskin</td>
			<td></td>
			<td></td>
			<td>De-identified human foreskin tissue for research purposes (Institutional Review Board- IRB #17574).</td>
		</tr>
		<tr>
			<td>KGM-GOLD keratinocyte medium (KGM gold and supplements)</td>
			<td>Lonza</td>
			<td>00192151 and 00192152</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse alpha-smooth muscle actin antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>14-9760-82</td>
			<td>Dilution (1:200)</td>
		</tr>
		<tr>
			<td>Mouse Cytokeratin14 antibody</td>
			<td>Abcam</td>
			<td>ab7800</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr>
			<td>Mouse S100 antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>MA5-12969</td>
			<td>Dilution (1:200)</td>
		</tr>
		<tr>
			<td>Multi chambered (4 well glass slide)</td>
			<td>Tab-Tek</td>
			<td>154526</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoCounter -Via1-Cassette</td>
			<td>Chemometec</td>
			<td>941-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysisne (PLL)</td>
			<td>ScienCell Research</td>
			<td>32503</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Anti-fade Mountant with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit K10 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4501656</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr>
			<td>Rabbit p75-NTR antibody</td>
			<td>Millipore</td>
			<td>AB1554</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr>
			<td>Rabbit vimentin</td>
			<td>ProteinTech</td>
			<td>10366-1-AP</td>
			<td>Dilution (1:200)</td>
		</tr>
		<tr>
			<td>Schwann cell culture medium</td>
			<td>ScienCell Research</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision tweezers DUMONT straight with extra fine tips Dumostar, 5</td>
			<td>ROTH</td>
			<td>LH75.1</td>
			<td>Sterilize with 70% alcohol before use</td>
		</tr>
		<tr>
			<td>IRIS Scissors, sharp/sharp. Length 4&#8211;3/8&#34;(111mm)</td>
			<td>Codman</td>
			<td>54-6500</td>
			<td>Sterilize with 70% alcohol before use</td>
		</tr>
		<tr>
			<td>Sterilized surgical - sharp blade (Duro Edge Economy Single Edge Blades)</td>
			<td>Razor blade company</td>
			<td>94-0120</td>
			<td>Sterilize with 70% alcohol before use</td>
		</tr>
		<tr>
			<td>T25 culture flask</td>
			<td>Corning</td>
			<td>353109</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin neutralization buffer (TNS)</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer 7- Axiocam 506 mono &#8211; Apotome.2 microscope</td>
			<td>For immunofluorescence of chamber slide containing stained cells</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>ZEISS</td>
			<td>Primovert</td>
			<td>For visulaizing/observing cell attachment or detachment</td>
		</tr>
	</tbody>
</table>

isolation, culture, and characterization of primary schwann cells, keratinocytes, and fibroblasts from human foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

Normal neonatal foreskin was used for the isolation of primary epidermal keratinocytes and dermal SCs and fibroblasts. The isolated primary cells were cultured in respective cell culture media containing growth factors. After seeding of SCs and fibroblast in culture flasks, most of the cells adhered to the bottom of the flask within 2 h. In the case of keratinocytes, most keratinocytes adhered by 24 h. Isolated epidermal primary keratinocytes reached 85% confluence by day 7 and exhibited characteristic cell morphology (c...

Watch this Scientific Journal Video about Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin at JoVE.com

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

isolation-culture-characterization-primary-schwann-cells

Research

JoVE Journal

Neuroscience

5.9K Views.  The Pennsylvania State University College of Medicine. The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin. 

Video: Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.