The authors wish to acknowledge Becky Marquez, the clinical coordinator at the Sentara Bariatric Center, for her assistance with the process of patient screening and consenting. This research was supported by R15HL114062 to Anca D. Dobrian.

The Institutional Review Board Committee at Eastern Virginia Medical School approved the research and collection of human adipose tissue samples used in the study. Informed written consent was collected from the patients.
1. Preparation of Buffers, Media, and Instruments
<ol>
	<li>Prepare a Krebs Ringer Bicarbonate-Buffered Solution (KRBBS): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1 mM adenosine, 0.01% antibiotic/antimycotic mix (50 &#181;g/mL of penicillin, 50 &#181;g/mL of streptomycin, 30 &#181;g/mL of gentamicin, 15 ng/mL of amphotericin) and 10 mM HEPES (pH = 7.4). This solution is prepared fresh each time for the tissue collection and digestion.</li>
	<li>Prepare a fresh collagenase solution: 1 mg/mL of collagenase, type 1, in KRBSS; make 3 mL/g of fat. Pre-warm the collagenase solution at 37 &#176;C in a water bath prior to the tissue digestion.</li>
	<li>Prepare a CD34 Cell Isolation Buffer: 2% fetal bovine serum (FBS) and 1 mM EDTA in sterile phosphate-buffered saline (PBS).</li>
	<li>Prepare Adipogenesis Media: DMEM/F12, 5% FBS, 50 &#181;g/mL of penicillin/streptomycin, 1.25 &#181;L/mL of human insulin (5 &#181;g/mL or 144 mU/mL), and 0.36 &#181;L/mL of 2.8 mM rosiglitazone (1 &#181;M).</li>
	<li>Prepare a 0.3% Oil Red O (ORO) stock solution (weight/volume) by dissolving 0.3 g of ORO into 100 mL of isopropanol.</li>
</ol>
2. Adipose Stromal Vascular Fraction Isolation
NOTE: The study included a cross-sectional cohort of morbidly obese type 2 diabetic (T2D) and non-diabetic subjects, aged 18&#8211;65 years, undergoing bariatric surgery at the Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, VA). Exclusion criteria included an autoimmune disease including type 1 diabetes mellitus, conditions requiring chronic immunosuppressive therapy, anti-inflammatory medications, thiazolinendiones, active tobacco use, chronic or acute infections, or a history of malignancy treated within the last 12 months. T2D was defined as a fasting plasma glucose of 126 mg/dL or greater, a glucose of 200 mg/dL or greater after a 2 h glucose tolerance test, or the use of antidiabetic medications.
<ol>
	<li>Collect human omental (OM) and subcutaneous (SC) adipose tissue (AT) from human subjects undergoing bariatric surgery.</li>
	<li>Keep the OM and SC AT in separate vials containing Hank&#8217;s Buffered Salt Solution with 50 &#181;g/mL of penicillin/streptomycin at room temperature immediately after tissue extraction.</li>
	<li>Carefully clean and remove fibrotic and cauterized sections of the tissue by using 70% ethanol swabbed scissors and tweezers when the sample arrives at the lab after the tissue extraction.</li>
	<li>Weigh the adipose tissue and partition it into 5 g (or less) aliquots for the collagenase digestion.</li>
	<li>Finely mince 5 g of the AT in 5 mL of a collagenase solution in a scintillation vial at room temperature, using two pairs of scissors.</li>
	<li>Add an additional 10 mL of collagenase solution to the minced tissue for 15 mL total.</li>
	<li>Digest the samples for 1 h, at 37 &#176;C, in a water bath with continuous shaking.</li>
	<li>Cut the tip off a 20 mL syringe to make a blunt end.</li>
	<li>Cut a 9 cm x 9 cm square from a 250 &#181;m mesh and push it halfway into a 50 mL conical tube using the blunt end syringe.</li>
	<li>Pour the samples into the 20 mL blunt end syringe and filter through the 250 &#181;m nylon mesh into the 50 mL conical tube to separate adipocytes and stromal vascular cells from undigested tissue. 
	NOTE: Do not use more than 5 g of AT per 50 mL conical tube, as the mesh will get clogged due to excess fibrotic undigested material.</li>
	<li>Wash out the scintillation vial with 10 mL of KRBBS and pour it through the filter to collect residual cells.</li>
	<li>Incubate the filtered samples for 5 min at room temperature. 
	Note: This step is important to allow for the adipocytes to float at the top of the tube and some of the stromal vascular cells to settle on the bottom of the tube.</li>
	<li>Utilize a 20 mL syringe and a 20 G x 6 in pipetting needle to remove the stromal vascular fraction layer and transfer it to a clean 50 mL conical tube.</li>
	<li>Add 10 mL of KRBBS and allow the samples to sit on the bench for 5 min, at room temperature.</li>
	<li>Repeat steps 2.12&#8211;2.14 twice. 
	Note: These steps will ensure that there is no contamination of the stromal vascular cells with the floating adipocytes.</li>
	<li>Keep the stromal vascular fractions from both depots on ice for further processing, as described in the steps below. 
	Note: Do not leave the cells on ice for more than 1 h, as this may have an impact on the cell viability. The adipocytes can be flash-frozen in liquid nitrogen for long-term storage or used fresh for experiments.</li>
</ol>
3. Isolation of Adipose Tissue Endothelial Cells
<ol>
	<li>Spin the stromal vascular fractions (SVF) at 500 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove the samples from the centrifuge and carefully pour off the supernatant; keep the pellet containing the SVF cells.</li>
	<li>Gently resuspend the cell pellets in 5 mL of PBS. 
	NOTE: If more than 5 g of an AT depot was processed into multiple aliquots (see step 2.4), pool the cell pellets together.</li>
	<li>Spin the samples at 500 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove the supernatant, resuspend the cells in 1 mL of PBS, and count the cells. 
	NOTE: Here, an automatic cell counter was used for cell counting. The cell viability was obtained by mixing 12 &#181;L of cell suspension with 12 &#160;&#181;L of an acridine orange/propidium iodide (AO/PI) solution (see Table of Materials). The average number of cells per gram of AT for each depot is: (a) OM Depot: 1.69 x 105 &#177; 4.51 x 104; (b) SC Depot: 1.24 x 105 &#177; 6.45 x 104. The viability of the cells from both depots was &#62;90%.</li>
	<li>Pellet the samples at 500 x g for 5 min, at 4 &#176;C. 
	NOTE: A CD34 immunomagnetic selection protocol (see Table of Materials) was adapted for steps 3.7&#8211;3.15.</li>
	<li>Resuspend the pellet in 100 &#181;L of CD34 isolation buffer. 
	NOTE: The total cell counts require the following re-suspension volumes: (a) &#60;2 x 107 cells: resuspend the pellet in 100 &#181;L; (b) 2 x 108&#8211;5 x 108 cells: resuspend the pellet in 1 mL. In steps 3.9&#8211;3.16, the volumes of the reagents used were scaled down for &#60;2 x 107 cells.</li>
	<li>Add 10 &#181;L of CD34 cocktail and incubate the sample for 15 min, at room temperature.</li>
	<li>Add 5 &#181;L of magnetic beads and incubate the sample for 10 min, at room temperature.</li>
	<li>Add 2.5 mL of CD34 isolation buffer to each sample and place the samples into the magnet, for 5 min, at room temperature.</li>
	<li>Invert the magnet for 5 s to pour off the isolation buffer.</li>
	<li>Remove the samples from the magnet and repeat steps 3.10 and 3.11 4x.</li>
	<li>Remove the tube from the magnet and add 2 mL of CD34 isolation buffer.</li>
	<li>Pellet the cells at 500 x g for 5 min, at 4 &#176;C.</li>
	<li>Resuspend the cell pellets in 1 mL of CD34 isolation buffer and count the cells. 
	NOTE: Here, the average number of cells per gram of AT is: (a) OM Depot: 4.75 x 104 &#177; 3.36 x 104; (b) SC Depot: 1.06 x 104 &#177; 9.53 x 103. A CD31 plastic immunobead protocol was adapted for steps 3.16&#8211;3.34 (see Table of Materials).</li>
	<li>Pellet cells at 500 x g for 5 min and resuspend the pellet in 250 &#181;L of Buffer B and 250 &#181;L of wash buffer.</li>
	<li>Thoroughly resuspend the CD31 beads by vortexing the tube.</li>
	<li>Add 20 &#181;L of CD31 beads. 
	NOTE: Due to total cell counts &#62;1 x 106, the volume was scaled down according to the manufacturer&#8217;s input.</li>
	<li>Incubate the sample with rocking for 30 min, at room temperature.</li>
	<li>Attach a strainer to a sterile 50 mL conical tube. 
	NOTE: The larger opening of the strainer must be on top.</li>
	<li>Prior to the cell separation, add 1 mL of wash buffer to equilibrate the strainer. Apply the mixture generated under step 3.16 to the strainer.</li>
	<li>Wash the strainer with 5 mL of wash buffer in a circular motion for a total volume of 20 mL. Attach the connector to a sterile 50 mL conical tube and close the luer-lock.</li>
	<li>Attach the strainer to the connector. Add 1 mL of wash buffer along the wall of the strainer. Add 1 mL of activated buffer D along the wall of the strainer. Swirl the sample gently and incubate it for 10 min, at room temperature.</li>
	<li>Add 1 mL of wash buffer. Separate the cells from the beads by pipetting up and down 10x. 
	NOTE: Avoid generating air bubbles.</li>
	<li>Open the luer-lock and allow the detached cells to flow into the 50 mL conical tube. Wash the strainer 10x with 1 mL of wash buffer each time.</li>
	<li>Discard the connector and strainer and centrifuge the samples at 300 x g for 10 min, at 4 &#176;C. Resuspend the cell pellet in 2 mL of complete EGM-2 Media for culture. 
	Note: The average number of cells per gram of AT at this point is: (a) OM Depot: 5.92 x 103 &#177; 4.27 x 103; (b) SC Depot: 4.12 x 103 &#177; 2.1 x 103. The viability of cells from both depots was over 90%.</li>
</ol>
4. Induction of Adipogenesis in Isolated Endothelial Cells
<ol>
	<li>Grow the cells in 6-well tissue culture-treated plates with complete EGM-2 media in a 37 &#176;C, 5% CO2 incubator. Replace the media every 3&#8211;4 days until the cells reach 80&#8211;90% confluence. 
	NOTE: The population doubling is between 2&#8211;3 days.</li>
	<li>Expand the cells by plating them on 100 mm Petri dishes and split the cells 1:3 once. At this stage, the cells are on passage 2.</li>
	<li>Count the cells using an automatic cell counter (see Table of Materials).</li>
	<li>Seed approximately 100&#8211;200,000 cells into 6-well plates ensuring duplicate wells for each depot and culture them in complete EGM-2 media for 2 days.</li>
	<li>Aspirate off any growth media and replace it with 2 mL of DMEM/F12 with 5% FBS and 50 &#181;g/mL of penicillin/streptomycin for the control cells and replace it with 2 mL of Adipogenesis media (see step 1.4) for the treatment cells.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified 5% CO2 incubator for 13 days, replacing the respective media every 3 to 4 days. 
	NOTE: Cells will begin to accumulate lipids after 4 days of treatment.</li>
	<li>Conduct ORO and Nile red staining according to published methods15-17.</li>
	<li>To isolate cells for an RNA extraction, remove the media from the 6-well induction plates and wash it with 1 mL of sterile PBS.</li>
	<li>Add 350 &#181;L of a guanidium thiocyanate-phenol-chloroform solution (see Table of Materials) to each well and incubate it at room temperature for 5&#8211;10 min.</li>
	<li>Scrape the cells off the plate using a cell scraper and transfer the cells to a 1.5 mL microcentrifuge tube. 
	Note: The samples can be stored in the -80 &#176;C freezer for RNA extraction and further processing at a later date.</li>
	<li>Extract mRNA from the samples using an adapted RNA extraction protocol (see Table of Materials).</li>
	<li>Perform a real-time polymerase chain reaction (RT-PCR) to assess the gene expression using the following probes: adiponectin, UCP-1, and CIDEA (see Table of Materials). 
	NOTE: Here, RT-PCR procedures were carried out using the following standard protocol: denaturation (95 &#176;C; 15 s), annealing, and extension (60 &#176;C; 1 min) for 40 cycles. The samples that expressed genes with CT values &#62;35 were considered undetectable.</li>
</ol>

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K., Sugii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+fatty+potentials+for+therapy.">Adipose-derived stem cells: fatty potentials for therapy.</a> The International Journal of Biochemistry &amp; Cell Biology. 45 (6), 1083-1086 (2013).</li><li>Tchkonia, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+two+human+preadipocyte+subtypes+with+distinct+capacities+for+replication,+adipogenesis,+and+apoptosis+varies+among+fat+depots.">Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots.</a> American Journal of Physiology-Endocrinoloy and Metabolism. 288 (1), E267-E277 (2005).</li><li>van de Vyver, M., Andrag, E., Cockburn, I. L., Ferris, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinedione-induced+lipid+droplet+formation+during+osteogenic+differentiation.">Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation.</a> Journal of Endocrinology. 223 (2), 119-132 (2014).</li></ol>

The authors have nothing to disclose.

The focus of this paper is to provide a methodology for the isolation, expansion and adipogenic induction of CD34+CD31+ endothelial cells from visceral and subcutaneous depots of human adipose tissue.
Methodologies have been reported for the isolation of endothelial cells from various vascular beds of rodents or humans that involve primarily techniques using CD31 antibodies either fluorescently labeled or coupled to magnetic beads18,19...

Recent evidence shows that both in mice and in humans, a subset of cells residing in the adipose tissue vasculature can be differentiated into either white or beige/brown adipocytes1,2,3. The phenotype of such cells is a subject of controversy, with evidence supporting endothelial cells, smooth muscle/pericyte, or a spectrum of intermediate phenotypes4,5,6,7. The scope of developing this methodology was to test the adipogenic potential of CD34+CD31+ endothelial cells isolated from different fat depots from obese humans. Other studies in the literature are focusing on the adipogenic potential of the total stromal vascular fraction or of the known adipocyte progenitors2,8,9. Since currently existing technologies can target specifically adipose tissue endothelial cells for drug delivery10, understanding the potential of such cells to undergo adipogenic induction towards white or beige adipocytes is important for future targeted therapies.
Different groups reported the combination of CD31 and CD34 markers as surrogates to isolate endothelial cells from human adipose tissue11,12,13. Typically, the isolation is performed using two sequential steps and a positive selection using magnetic beads. In this report, immunoseparation using CD34+ magnetic beads combined with CD31 plastic beads was utilized. We found this technique superior to the sequential magnetic immunoseparation with respect to the preservation of typical cobblestone endothelial morphology. Also, we were able to generate enough cells required for the expansion and adipogenic induction starting from as little as 1&#8211;2 g of fat. A small sample biopsy of subcutaneous fat is enough to produce the required quantity of cells for downstream applications. This aspect is potentially important, particularly if this method will be utilized for screening for a responsiveness to adipogenic induction in human subjects.
Unlike other systems reported in the literature, this method utilizes only two ingredients for the adipogenic induction of the CD34+CD31+ cells: a PPAR&#947; agonist&#8212;rosiglitazone&#8212;and human insulin. Importantly, the amount of insulin used falls within the normal/high range of circulating post-absorptive insulin in humans14. The degree of responsiveness to insulin of the cells in vitro, measured by Akt phosphorylation, does not correlate with their ability to respond to the induction cocktail. Interestingly, using this induction cocktail and experimental conditions, a mix of white and beige/brown cells were obtained as determined by the size and numbers of intracellular lipid droplets and the expression of molecular markers. This straightforward and cost-effective induction protocol along with the quantitative evaluation of the phenotype of the responder cells (white vs. beige) allows for a screening of agents that can potentially alter the balance of differentiated beige:white adipocytes.
This method also provides a translational approach for understanding the underlying mechanisms of adipogenesis of vascular endothelial progenitors in human adipose tissue. Using this specific isolation/differentiation technique, investigators can interrogate various pathways responsible for adipogenesis in a subset of vascular endothelial cells from various fat depots in lean and obese humans.

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

Our protocol aims to provide an in vitro approach to determine the adipogenic potential of CD34+CD31+ vascular cells from different depots of human adipose tissue. A simplified flowchart diagram is shown in Figure 1A. The first step using a positive selection of CD34 expressing cells results in &#62; 95% CD34+ cells in the population of the freshly isolated cells (Figure 1A). Importantly, this marker is lost after the ce...

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

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Immunology and Infection

11.7K Views.  Eastern Virginia Medical School. The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Video: Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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 ...