We were supported with expert technical assistance by the HSCI-CRM Flow Cytometry facility at Massachusetts General Hospital and the Bauer Core Facility at Harvard University. T.K and K. G were supported by the Swedish Research Council and C.M. by the German Research Foundation. We thank Sergey Isaev and I-Hsiu Lee for assistance in analysis of single-cell RNA sequencing data.

All work with human tissue was conducted after approval by the Massachusetts General Hospital Internal Review Board (IRB). All animal procedures were conducted in accordance with the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) guidelines. C57Bl/6 mice, 8-10 weeks old, and both males and females, were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Preparation of murine thymic tissue
<ol>
	<li>Prepare the following buffers and solutions.
	<ol>
		<li>Medium 199 (M199) with 2% (v/v) fetal bovine serum (FBS) (see Table of Materials).</li>
		<li>Murine dissociation cocktail: 0.5 WU/mL Liberase TM and 6.3 U/mL DNase I in M199 + 2% FBS (see Table of Materials).</li>
		<li>M199 with 2% (w/v) Bovine serum albumin (BSA). 
		NOTE: FBS containing medium can be switched to BSA to improve the RNA quality.</li>
	</ol>
	</li>
	<li>Perform the murine thymus dissection
	<ol>
		<li>Euthanize the mouse by CO2 asphyxiation (following institutionally approved protocols) and place it on its back. Wet the fur on the chest by spraying 70% ethanol and proceed to open the chest cavity.</li>
		<li>Carefully dissect the thymus by first making a transverse incision just below the rib cage with surgical scissors. Cut the ribs on each side of the mouse all the way up to the clavicle. Then cut the diaphragm so that the chest wall can be released. Lift the ribs with a pair of forceps to expose the thoracic cavity.
		<ol>
			<li>The thymus is a white, bilobular organ, sitting at the top of the cavity, just above the heart. Hold the thymus with the forceps and gently cut the connective tissue that holds the thymus in place. Put the dissected thymic tissue in the well of a 6-well plate with M199 + 2% FBS on ice. 
			NOTE: When working with aged or radiation-conditioned mice or mutant strains with thymic defects, the thymus may be so small that a dissection microscope is required to reliably excise the organ.</li>
		</ol>
		</li>
		<li>Using a dissection microscope, remove any extrathymic tissue using blunt-ended forceps and micro-spring scissors.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of murine thymic tissue (30 min in increments of 10 min)
	<ol>
		<li>Place the cleaned thymus in the cap of a 15 mL tube and finely mince the tissue with sterile surgical scissors. 
		NOTE: Mincing the tissue in the cap of the tube that is to be used for the digestion minimizes tissue loss due to transfer from one container to another. This is very important when working with aged or radiation-conditioned mice or mutant strains with thymic defects. The small thymus size in these settings necessitates the need to reduce the loss of material wherever possible.</li>
		<li>Add 2 mL of murine dissociation cocktail to the 15 mL tube, attach the cap containing the thymus pieces, and invert 5 times to ensure that the tissue is resuspended in the dissociation cocktail.</li>
		<li>To prevent leakage, wrap the cap of the tube in paraffin film. Then place the tube horizontally in a 37 &#176;C water bath with 250 rpm shaking. Incubate for 10 min. 
		NOTE: The success of the digestion protocol depends on the incubation being performed at 37 &#176;C and with agitation. The described protocol has been optimized using a shaking water bath. If that is not available, other approaches that enable agitation in a heated setting might work, but we recommend that the protocol be optimized for these settings first.</li>
		<li>Place the 15 mL tube in a rack and let the tissue pieces settle. Carefully remove the supernatant and pass it over a 70 &#181;m cell strainer into an ice-cold 50 ml tube.</li>
		<li>Repeat steps 2-4 twice for a total of 3 rounds of digestion. The tissue should be more or less completely dissociated by the end of the last incubation.</li>
		<li>Add 20 mL of M199 + 2% FBS to the collected cell suspension. Spin down at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend in an appropriate volume of M199 + 2% FBS. Then proceed to cell counting. 
		NOTE: The volume of M199 + 2% FBS to resuspend in will depend on the state of the thymus. For 6-10-week-old mice, we recommend resuspending the tissue in 3-4 mL of M199 + 2% FBS. However, for experiments using aged or radiation-conditioned tissue or mutant strains with thymic defects, the small thymus size will require lower volumes ranging from 0.1-1 mL.</li>
		<li>Count cells by diluting the cell suspension 1:2 in 0.4% (w/v) trypan blue solution. Load cells onto a hemocytometer and count under a bright-field microscope. Count unstained cells to determine the number of live cells per milliliter and the number of blue-stained cells to assess sample viability.</li>
	</ol>
	</li>
	<li>Perform fluorescence activated cell sorting of murine thymic stroma
	<ol>
		<li>Spin down the cells at 500 x g for 5 min at 4 &#176;C. Resuspend cells at a concentration of 5 x 107 cells/mL in M199 + 2% FBS supplemented with 25 U/mL Protector RNase inhibitor (see Table of Materials).</li>
		<li>Add murine Fc Block (see Table of Materials) at a concentration of 2 &#181;g/mL and incubate for 10 min at 4 &#176;C.</li>
		<li>Stain the cells with the following antibodies: CD45-PE/Cy7 (4 &#181;g/mL), Ter119-PE (4 &#181;g/mL), CD31-BUV737 (2 &#181;g/mL), and EpCam-BV711 (2 &#181;g/mL) (see Table of Materials) for 30 min at 4 &#176;C.</li>
		<li>Wash the samples in M199 + 2% BSA and spin down at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove supernatant and resuspend cells in M199 + 2% BSA containing a viability dye such as DAPI (0.5 &#181;g/mL) or 7-AAD (1 &#181;g/mL) (see Table of Materials). Proceed to FACS isolation of thymic stromal cells following established protocols19.</li>
	</ol>
	</li>
</ol>
2. Preparation of human thymic tissue
<ol>
	<li>Prepare the following buffers and solutions.
	<ol>
		<li>Medium 199 (M199) with 2% (v/v) FBS.</li>
		<li>M199 with 1.5% (w/v) BSA.</li>
		<li>Human dissociation cocktail: 1 mg/ml Collagenase IV and 2 mg/mL Dispase and 6.3 U/mL DNase I in M199 +1.5% BSA (see Table of Materials). 
		NOTE: The last digestion step in the preparation of human thymic tissue includes trypsin. It is therefore essential that all steps up until that point are carried out in BSA containing buffer as FBS will inhibit trypsin activity.</li>
	</ol>
	</li>
	<li>Perform human thymic tissue isolation
	<ol>
		<li>Place the human thymus (collected following previously published method19) in a suitable volume of M199 + 1.5% BSA and store it on ice until processing starts.</li>
		<li>While working with human thymic tissue, handle it as if it is potentially infectious by carrying out the work in a tissue culture hood and using standard precautions per CDC guidelines20.</li>
		<li>Put the tissue in a 10 cm Petri dish with 10 mL of ice-cold M199 + 1.5% BSA. Using sterile forceps and scissors, carefully clean away extrathymic fat or tissue that was damaged as the organ was extracted.</li>
		<li>Using surgical scissors and a pair of forceps, cut the thymus into 1 cm3 cubes and use the plunger of a 10 mL syringe to gently push down on the pieces. This will release some of the developing thymocytes, thus reducing the volume of tissue to be digested. 
		NOTE: Save the released hematopoietic fraction if subsequent analysis of thymocyte developmental stages is needed. However, do not combine this fraction with the stromal cell fraction generated during enzymatic digestion. It was observed that pooling the two fractions significantly increases the risk of clogs forming in the cell suspension, ultimately reducing the yield of stromal cells. Few stromal cells are lost during the gentle crushing of the tissue.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of human thymic tissue (90 min in increments of 30 min)
	<ol>
		<li>Transfer approximately 5 mg of the lightly crushed thymic tissue pieces to the cap of a 50 mL conical tube. Then cut the tissue finely using sterile surgical scissors.</li>
		<li>Pipette 8 mL of human dissociation cocktail into the 50 mL tube, attach the cap containing the minced human thymic tissue, and then gently invert the tube 5 times to ensure that all the tissue is mixed in with the enzyme cocktail.</li>
		<li>Secure the cap of the tube with paraffin film to prevent leakage. Incubate the tube horizontally in a shaking (250 rpm) water bath at 37 &#176;C for 30 min.</li>
		<li>Place the 50 mL tube in a rack and let the human thymic tissue pieces settle before removing the supernatant and passing it over a 70 &#181;m cell strainer into a new 50 ml tube placed on ice.</li>
		<li>Repeat steps 2.3.2-1.3.4 for a total of two rounds of digestion with the human dissociation cocktail.</li>
		<li>After the second round of digestion is done, add an additional 8 mL of the human dissociation cocktail to the remaining tissue. Then also pipette 2 mL of 0.25% trypsin into the tissue dissociation tube. Incubate for an additional 30 min at 37 &#176;C, shaking at 250 rpm. 
		NOTE: It is absolutely essential that the digestion time for human tissue is at least 90 min, as shorter incubation times lead to drastically reduced stromal cell yields.</li>
		<li>Once the last incubation step is done, pool the cell suspension and remaining tissue fragments with the supernatant collected in previous steps by passing over a 70 &#181;m cell strainer. Add 3 mL of FBS to the sample to break the trypsin reaction and place on ice.</li>
		<li>Proceed to counting the cells as described in step 1.3.</li>
	</ol>
	</li>
	<li>Perform fluorescence activated cell sorting of human thymic stroma
	<ol>
		<li>Spin down the cell suspension of human thymus and resuspend at a concentration of 5 x 107 cells/mL in M199 + 2% FBS supplemented with 25 U/mL Protector RNase inhibitor.</li>
		<li>Incubate with human Fc Block (5 &#181;g/mL) (see Table of Materials) for 10 min at 4 &#176;C.</li>
		<li>Stain the sample for 30 min at 4 &#176;C with CD45-BV711 (2.5 &#181;g/mL), CD235a-BV711 (2.5 &#181;g/mL), Lineage-cocktail-FITC (3.76 &#181;g/mL), CD66b-FITC (20 &#181;L/100 &#181;L sample), CD8-APC/Cy7 (5 &#181;L/100 &#181;L sample), CD4-BV605 (5 &#181;L/100 &#181;L sample), CD31-PE/Dazzle594 (10 &#181;g/mL), and EpCam-BV421 (2.5 &#181;g/mL) (see Table of Materials).</li>
		<li>Wash the samples in M199 + 1.5% BSA and spin down at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend cells in M199 + 1.5% BSA containing a viability dye such as DAPI (0.5 &#181;g/mL) or 7-AAD (1 &#181;g/mL). Proceed to FACS isolation of thymic stromal cells following established protocols19.</li>
	</ol>
	</li>
</ol>
3. Preparation of murine bone and bone marrow tissue
NOTE: Bone and bone marrow fractions are prepared in two separate digestion reactions to obtain maximum purity of stromal cells and optimal dissociation of tissues. The samples can be pooled after the digestion steps to be sorted as one stromal compartment.
<ol>
	<li>Prepare the following buffers and solutions.
	<ol>
		<li>M199 with 2% (v/v) FBS.</li>
		<li>Murine B&#38;M (bone and marrow) dissociation cocktail: Stemxyme 2 mg/mL, Dispase 1 mg/mL and 10 U/mL DNase I (see Table of Materials) in M199 +2% FBS. 
		NOTE: Prepare B&#38;M dissociation mix fresh and keep mix on ice until use.</li>
		<li>M199 with 0.5% (w/v) BSA.</li>
		<li>Phosphate-buffered saline (PBS) with 0.5% (w/v) BSA.</li>
		<li>M199 with 2% (w/v) Bovine serum albumin (BSA) and 2 mM EDTA. 
		NOTE: FBS containing medium can be switched to BSA to improve RNA quality.</li>
	</ol>
	</li>
	<li>Perform murine bone marrow separation
	<ol>
		<li>Euthanize the mouse by CO2 asphyxiation (following institutionally approved protocols). Spray with 70% ethanol to wet the fur.</li>
		<li>Carefully remove the femur, tibia, pelvis, and humerus from each side and place them in a well containing M199 + 2% FBS on ice21,22.</li>
		<li>Remove all muscle, ligament, and cartilage tissue with tissue wipes to minimize damage to the bone and preserve the periosteal layer. Place them in a new well with M199 + 2% FBS on ice.</li>
		<li>Use a scalpel and cut through the growth plates of the long bones to remove the epiphysis and place it in a 10 cm dish with M199 + 2% FBS on ice.</li>
		<li>Flush the bone marrow out of the bones until pale white. Use a 28 G needle and a 10 mL syringe filled with 10 mL M199 + 2% FBS to flush the marrow into a 15 mL tube on ice. Place the flushed bone in the 10 cm dish with the epiphysis. 
		NOTE: Complete flushing of the bone marrow for entire separation of tissues will result in better sample preparation.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of murine bone marrow tissue (total 30 min in increments of 10 min)
	<ol>
		<li>Leave the 15 mL tube standing upright on ice until the marrow sinks to the bottom of the tube. Slowly remove the 10 mL M199 + 2% FBS by pipetting.</li>
		<li>Add 4 mL of B&#38;M dissociation cocktail to the 15 mL tube and place it in a 37 &#176;C water bath (no shaking) for 10 min.</li>
		<li>After 5 min, invert the tube three times and place it back in the water bath.</li>
		<li>After the first 10 min, ensure the pellet is at the bottom and collect the supernatant while filtering through a 70 &#181;m cell strainer into an ice-cold 50 mL tube. Add 2 mL of fresh B&#38;M dissociation cocktail to the remaining BM pellet and place it back in the water bath.</li>
		<li>Dissociate any remaining bone marrow pieces by vigorous pipetting using a 1 mL pipette. Collect all the resulting cell suspension in the same tube as in the previous steps. Add 20 mL of M199 + 0.5% BSA + 2 mM EDTA to the collected cell suspension. 
		NOTE: In step 3.3.5, the buffer added after the final digested sample contains EDTA to aid in inhibiting further enzymatic activity.</li>
	</ol>
	</li>
	<li>Perform hematopoietic cell depletion of murine bone marrow tissue.
	<ol>
		<li>Centrifuge cells at 500 x g for 5 min at 4 &#176;C. Resuspend in 500 &#181;L of PBS + 0.5% BSA with biotin antibodies (5 mg/mL) targeting CD45, Ter119, CD11b, Gr1, B220, and CD3 (see Table of Materials). Transfer to a round-bottom tube with a cap.</li>
		<li>Incubate on an orbital shaker for 10 min at room temperature in the dark.</li>
		<li>Vortex the magnetic beads thoroughly and add 25 &#181;L of magnetic beads to the tube.</li>
		<li>Incubate on an orbital shaker for 5 min at room temperature in the dark.</li>
		<li>Place the tube in the matching magnet for 2-3 min and collect the supernatant in a fresh tube. 
		NOTE: The bone marrow can be pooled or kept separate from the bone fraction after this step. If the lineage depletion is successful, the resulting cell suspension should be devoid of red cells.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of murine bone tissue.
	<ol>
		<li>Break the bones into smaller pieces in the 10 cm dish with the flat cap-end of a 50 mL tube. Filter the supernatant through a 70 &#181;m cell strainer to isolate bone fragments inside the filter. 
		NOTE: Alternatively, a mortar and pestle can be used for bone fragmentation. This option, however, tends to generate more debris and lower stromal cell viability when the bone is ground rather than broken. The stroma cells are still attached to and in the bone, and the supernatant containing hematopoietic cells is to be discarded.</li>
		<li>Use scissors to cut the bone fragments in the cell strainer. This will increase the surface area for a more efficient enzymatic dissociation. Wash the bone with 5 mL of M199 + 2% FBS.</li>
		<li>Place the bone fraction in 5 mL of murine B&#38;M dissociation (see Table of Materials) mix in a new 50 mL tube.</li>
		<li>To prevent leakage, wrap the cap of the tube in paraffin film. Then place the tube horizontally in a 37 &#176;C water bath with 120 rpm shaking for 30 min.</li>
		<li>After the digestion, add 25 mL of M199 + 0.5% BSA + 2 mM EDTA and filter the supernatant containing the stromal cells through a 70 &#181;m cell strainer into an ice-cold 50 ml tube. 
		NOTE: Check that the resulting bone fragments have been visibly diminished by the digestion. In step 3.5.5, the buffer added after the final digested sample contains EDTA to aid in inhibiting further enzymatic activity.</li>
	</ol>
	</li>
	<li>Perform fluorescence activated cell sorting of murine bone and bone marrow stroma.
	<ol>
		<li>Spin down the cells at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend in murine Fc Block at a concentration of 10 &#181;g/mL in PBS + 0.5% BSA + 2 mM EDTA and incubate for 10 min at 4 &#176;C.</li>
		<li>Stain the cells (up to 10 million cells/100 &#181;L total staining volume) with the following antibodies: CD45-PE/Cy7 (1 &#181;g/mL), Ter119-PE/Cy7 (1 &#181;g/mL), CD31-BV421 (0.66 &#181;g/mL), CD140a-APC (2 &#181;g/mL), Sca1-AF700 (0.66 &#181;g/mL), CD51-PE (2 &#181;g/mL), CD105-PE/dazzle (2 &#181;g/mL) (see Table of Materials).</li>
		<li>Leave Calcein-AM to equilibrate to room temperature and resuspend in 50 &#181;L of DMSO. Prepare fresh for every use. Keep at room temperature after resuspension. Pre-dilute the calcein in PBS (0.1 mg/mL) before adding to the sample (20 &#181;g/mL) already resuspended in the antibody staining cocktail and incubate for 20-30 min at 4 &#176;C in the dark.</li>
		<li>Wash the samples in PBS + 0.5% BSA + 2 mM EDTA and centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend the cells in PBS + 0.5% BSA + 2 mM EDTA. Proceed to FACS isolation of bone and bone marrow stromal cells following established protocols19.</li>
	</ol>
	</li>
</ol>

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

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(110), e53936 (2016).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+compact+bone.">A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.</a> Nat Protoc. 5 (3), 550-560 (2010).</li><li>Calvi, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kode, A., 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=Leukaemogenesis+induced+by+an+activating+beta-catenin+mutation+in+osteoblasts.">Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts.</a> Nature. 506 (7487), 240-244 (2014).</li><li>Agarwal, P., 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=Mesenchymal+niche-specific+expression+of+cxcl12+controls+quiescence+of+treatment-resistant+leukemia+stem+cells.">Mesenchymal niche-specific expression of cxcl12 controls quiescence of treatment-resistant leukemia stem cells.</a> Cell Stem Cell. 24 (5), 769-784 (2019).</li><li>Duarte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+endosteal+vascular+niche+remodeling+rescues+hematopoietic+stem+cell+loss+in+aml.">Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in aml.</a> Cell Stem Cell. 22 (1), 64-77 (2018).</li><li>O'flanagan, C. H., 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=Dissociation+of+solid+tumor+tissues+with+cold+active+protease+for+single-cell+rna-seq+minimizes+conserved+collagenase-associated+stress+responses.">Dissociation of solid tumor tissues with cold active protease for single-cell rna-seq minimizes conserved collagenase-associated stress responses.</a> Genome Biol. 20 (1), 210 (2019).</li><li>Stoeckius, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+hashing+with+barcoded+antibodies+enables+multiplexing+and+doublet+detection+for+single+cell+genomics.">Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.</a> Genome Biol. 19 (1), 224 (2018).</li></ol>

D.T.S. is a director and shareholder for Agios Therapeutics and Editas Medicines; a founder, director, shareholder, and scientific advisory board member for Magenta Therapeutics and LifeVault Bio, a shareholder and founder of Fate Therapeutics and Garuda Therapeutics, and a director, founder, and shareholder for Clear Creek Bio, a consultant for FOG Pharma, Inzen Therapeutics and VCanBio, and a recipient of sponsored research funding from Sumitomo Dianippon. D.T.S and K.G are inventors of patent US20220143099A1.

Stromal cells in hematopoietic organs are critical for normal blood production and hematopoietic stroma perturbations can result in severe impairments in hematopoietic maintenance and response to stress9,23,24. Insight into hematopoietic stromal cells is essential for understanding hematological diseases7,9,10,<sup class="xre...

In the healthy adult, de novo production of blood cells occurs in the bone marrow and the thymus. Stromal cells at these sites are essential for maintenance of hematopoiesis, but stroma constitutes less than 1% of the tissue1,2,3,4. Obtaining pure isolates of hematopoiesis supporting stroma therefore constitutes a significant challenge, particularly for single-cell multiomics that requires expedient processing to obtain samples of high quality. Components of different digestion cocktails may interfere with certain steps in multiomics analysis5,6. The protocols presented here detail the isolation of a wide variety of stromal cells from bone marrow and thymic tissues.
Perturbations of stromal constituents in both bone marrow and thymus result in profound disruption in blood cell development and can result in malignancies7,8,9. Hematopoiesis supporting stroma is damaged following cytotoxic conditioning and bone marrow transplantation, resulting in reduced secretion of cytokines and growth factors that sustain hematopoietic stem and progenitor cells (HSPCs)2,10,11. Furthermore, aging affects bone marrow and thymus stromal cells likely contributing to aged hematopoietic phenotypes. The thymus is the first organ to undergo extensive age-associated involution. Fat and fibrotic tissue start replacing T cell supportive stroma as early as the onset of puberty12,13. In the bone marrow, adipocyte content increases with age and the vascular and endosteal niches are significantly remodeled14,15,16.
To enable study of hematopoiesis supportive stroma across multiple stress states and in the case of the thymus of both human and murine tissue, we have optimized previously published digestion protocols1,2,8,17,18. These protocols ensure efficient and reproducible isolation of cells, and they are compatible with single-cell RNAsequencing (scRNAseq) and other types of multiomics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>7AAD (7-aminoactinomycin D)</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human Lineage Cocktail 3-FITC</td>
			<td>BD Biosciences</td>
			<td>643510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl/6 mice</td>
			<td>Jackson</td>
			<td>664</td>
			<td>Males or females, 8-12 weeks old</td>
		</tr>
		<tr>
			<td>Calcein&#160;</td>
			<td>Fisher Scientific</td>
			<td>65-0853-78</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Millipore Sigma</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Sterile Cell Strainers, White, Mesh Size: 70 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Biolegend</td>
			<td>422801</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Thermo Fisher Scientific</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>90083</td>
			<td>&#160;2500 U/mL</td>
		</tr>
		<tr>
			<td>Easysep mouse streptavidin RapidSpheres Isolation kit</td>
			<td>StemCell Technologies</td>
			<td>19860</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A31605-01</td>
			<td>Qualified One Shot</td>
		</tr>
		<tr>
			<td>Human Fc Block</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM&#160;</td>
			<td>Millipore Sigma</td>
			<td>5401127001</td>
			<td>Research Grade</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>12350</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD235a-BV77</td>
			<td>BD Biosciences</td>
			<td>740785</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD31-PE/Dazzle594</td>
			<td>Biolegend</td>
			<td>303130</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD45-BV77</td>
			<td>Biolegend</td>
			<td>304050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD4-BV605</td>
			<td>BD Biosciences</td>
			<td>562658</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD66b-FITC</td>
			<td>BD Biosciences</td>
			<td>555724</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD8-APC/Cy7</td>
			<td>BD Biosciences</td>
			<td>557760</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human EpCam-BV421</td>
			<td>Biolegend</td>
			<td>324220</td>
			<td></td>
		</tr>
		<tr>
			<td>Protector RNase Inhibitor</td>
			<td>Millipore Sigma</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD105-PE /dazzle594</td>
			<td>Biolegend</td>
			<td>120424</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD11b-Biotin</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD140a-APC</td>
			<td>Fisher Scientific</td>
			<td>17-1401-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD31-BUV737</td>
			<td>BD Biosciences</td>
			<td>612802</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD31-BV421</td>
			<td>Biolegend</td>
			<td>102424</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD3-Biotin</td>
			<td>Biolegend</td>
			<td>100244</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45.2-Biotin</td>
			<td>Biolegend</td>
			<td>109804</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45-PE/Cy7</td>
			<td>Biolegend</td>
			<td>103114</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45-PE/Cy7</td>
			<td>Biolegend</td>
			<td>103114</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45R/B220-Biotin</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD51-PE</td>
			<td>Biolegend</td>
			<td>104106</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse EpCam-BV711</td>
			<td>BD Biosciences</td>
			<td>563134</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly-6A/E(Sca-1)-AF700</td>
			<td>Biolegend</td>
			<td>108142</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ter119-Biotin</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ter119-PE</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ter119-PE/Cy7</td>
			<td>Biolegend</td>
			<td>116222</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemxyme&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004107</td>
			<td></td>
		</tr>
	</tbody>
</table>

stromal cell isolation from hematopoietic organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

These protocols yield reproducible stromal cell varieties from the thymus and bone marrow suitable for flow cytometric analysis, as well as single-cell multiomics, such as scRNA sequencing. Murine thymic tissue undergoes significant remodeling in response to stressors, such as the cytotoxic conditioning that precedes bone marrow transplantation or the natural aging process. As a consequence, thymic cellularity is drastically reduced in both of these settings (Figure 1A). While a thymus from ...

Watch this Scientific Journal Video about Stromal Cell Isolation From Hematopoietic Organs at JoVE.com

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

stromal-cell-isolation-from-hematopoietic-organs

Research

JoVE Journal

Immunology and Infection

965 Views.  Massachusetts General Hospital. Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Video: Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...