The authors gratefully acknowledge the Flow Core Facility and the Glasgow Imaging Facility (GIF) within the School of Infection and Immunity for their support and assistance in this work.

The authors declare that they have no competing interests.

The easy culture and isolation of large numbers of functional OCs in vitro are important for advancing the understanding of bone biology and OC-mediated diseases. Classically, OCs were generated in co-cultures with osteoblasts or stromal cells and hematopoietic cells from the spleen or bone marrow38,39. A significant breakthrough in the understanding of osteoclastogenesis was the identification of RANKL as the major regulator of OC formation, differentiation, and survival40. Early protocols of RANKL-dependent culture systems utilized PBMCs for OC generation21,41,42. However, these mixed cultures are lengthy and present many confounding factors that limit the ability to test the direct effects on OC differentiation and function. This protocol describes an efficient and reliable in vitro model of osteoclastogenesis from human peripheral CD14+ monocytes in which optimal osteoclastogenesis can be obtained within 7 days (Figure 1 and Figure 2), which is considerably faster when compared to some other protocols43,44,45,46. The main distinguishing features of this protocol are (1) the use of purified CD14+ monocytes, (2) the priming of the monocytes with M-CSF prior to exposure to RANKL, (3) the length of the culture (&#60;7 days), and (4) the reliable detection of the inhibition of OC formation (TRAP staining) and function (resorption, ATP production, actin ring reorganization) with inhibitors. 
During the optimization of the methodology, several critical points were identified. It has been observed that the in vitro differentiation of OCs is largely dependent on the seeding density of the CD14+ monocytes. Thus, in this protocol, the cells are seeded at a high density (1 x 105 cells/well of a 96-well plate, in 100 &#181;L of medium), as it is essential for the cells to be able to interact with each other and to be in proximity to fuse and become mature OCs. Similarly, seeding cells at a density that is too high limits their differentiation and growth due to medium limitations and a lack of the required space. Furthermore, to achieve maximum success with this protocol, it is important to perform the density gradient separation carefully and to ensure that the enriched population of CD14+ cells is as pure as possible. For example, inadequate washing steps result in a lack of removal of platelets, which consequently inhibits OC differentiation47,48. Similarly, the presence of minor T cell contamination in isolated CD14+ preparations stimulated with M-CSF alone can result in OC differentiation, potentially via RANKL secretion by T cells49. Therefore, it is important to include an M-CSF control for every experiment. A routine purity check, especially when using a new isolation kit, is also recommended to ensure the purity of the sample.
Optimum OC numbers (range: ~200-1,600 OCs/well) are achieved using &#945;-MEM medium enriched with nucleosides and L-glutamine. Other conventional culture media, including Dulbecco&#39;s modified eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium, affect the OC yield. The source of FBS can also influence osteoclastogenesis. Different batches of FBS can lead to reduced RANK-L-derived osteoclastogenesis, as well as the appearance of low numbers of TRAP+ multinucleated cells in the M-CSF controls (Supplementary Figure 3). Therefore, to achieve consistent results, it is advised to test new FBS batches before use and to continue with the same batch throughout the experiments to minimize variations in the differentiation process. Additionally, donor-to-donor variability, in terms of total numbers of differentiated OCs obtained at the end time point, constitutes a limitation when using this protocol to compare, for instance, healthy donors to patients. In these cases, it is imperative to use exactly the same conditions and the same lot of medium, FBS, and other reagents.
Another necessary step for optimum OC differentiation and maturation is priming the monocytes with M-CSF before the RANKL addition. The exposure of the cells to M-CSF 18-24 h prior to RANKL primes the monocytes to upregulate RANK expression15,26. The addition of RANKL at this time point ensures optimal OC differentiation in a dose-dependent manner. The degree of OC differentiation varies from donor to donor; however, 25 ng/mL RANKL is usually sufficient to differentiate a high number of OCs in most donors. Additionally, 25 ng/mL RANKL can be used in assays for the initial screening of compounds, as it facilitates the evaluation of both the enhancing and inhibitory effects of the test compounds. Other culture systems have used longer M-CSF pre-incubation times prior to RANKL addition, but this results in a longer culturing time for osteoclastogenesis50. In addition, leaving the primed monocytes to incubate overnight allows them to attach to the plate, albeit not in a fully adherent state. Therefore, when RANKL is introduced for the first time, the medium must be half-changed very carefully rather than completely changed to prevent the detachment and loss of the primed monocytes. The medium also needs to be refreshed every 3-4 days to avoid medium depletion and prevent cell death. Moreover, due to the low volume used in this assay (100 &#181;L/well in a 96-well plate), it is of the utmost importance to have a frame of empty wells that are filled with an aqueous solution (i.e., sterile distilled H2O or PBS) around the assay wells. This prevents medium evaporation and edge effects.
Finally, for metabolic assays (e.g., ATP assays), it is imperative that the cells are viable to avoid huge standard deviation between replicates (Figure 5). High viability of the cells is also important for sorting the cells and for the further culturing of the sorted OCs (Figure 4). This method, however, has several limitations. Fully mature OCs are very adherent and difficult to detach from the plates. The larger OCs are often impossible to detach, which can lead to a lower cell yield. Therefore, the cells need to be counted after sorting and prior to plating at the required concentration. Furthermore, in the present protocol, a non-enzymatic method (accutase) to detach the OCs is used to prevent membrane alterations in downstream surface staining for flow cytometry. The use of cell scrapers (both with soft or hard endings) was also tested and led to high cell death. Enzymatic detachment using 0.05% Trypsin/EDTA solutions can be used for a higher yield of detached OCs when membrane integrity is not required for downstream applications. Additionally, to prevent the OCs from clumping together, the use of a high concentration of EDTA in all the buffers following cell detachment, as well as appropriate filtering prior to flow cytometry acquisition, are highly recommended. It is important to note that OC cultures are a heterogeneous population of cells consisting of mature OCs, OC precursors, and macrophages. Macrophages can be easily distinguished from OCs, although both mononuclear pre-OCs and multinuclear OCs express OSCAR and cannot be distinguished with the present method (Figure 4). Indeed, this latter issue constitutes the main limitation of this method. In addition, a low expression of OSCAR is also present in M-CSF cultures (Figure 4B) and might indicate macrophages that are primed for OC lineage commitment. It is important to set the gate for OSCAR+ cells based on the FMO staining signal, as shown in Figure 4B.
In summary, this protocol describes an optimized and robust method for the efficient production of active and functionally mature OCs from circulating primary human monocytes. The strength of this protocol is its ability to generate OCs in a short time duration and yield high numbers of differentiated OCs. This method opens the way for investigating the basic mechanisms underlying OC differentiation and function.

Osteoclasts (OCs) are multinucleated giant cells of hematopoietic lineage with a unique capacity to resorb bone. They are responsible for the development and continuous remodeling of the skeleton1,2. In the skeletal phases of development, OCs and tissue-resident macrophages are derived from erythro-myeloid progenitors and colonize the bone niche and organ tissues. In physiological conditions, erythro-myeloid progenitors are required for normal bone development and tooth eruption, while the influx of circulating blood monocytes into the bone niche provides postnatal maintenance of the OCs, bone mass, and bone marrow cavity3. Under pathological conditions, monocytes are recruited to sites of active inflammation and can contribute to pathological bone destruction4,5.
Patients with several forms of arthritis experience joint inflammation, leading to progressive joint destruction caused by OCs6. For instance, in rheumatoid arthritis (RA), overactivated OCs are responsible for pathological bone erosion and joint destruction7,8, and current treatments often do not improve or stop the bone damage9,10,11. Alterations in circulating monocytes both in terms of population distribution and transcriptomic and epigenetic signatures have been reported in RA patients12,13,14. Moreover, it has been reported that altered monocyte responses to inflammatory stimulation affect osteoclastogenesis in RA patients with active disease15,16,17.
The differentiation of OCs is a complex multistep process comprising the commitment of myeloid precursor cells to differentiation into OC precursors. During osteoclastogenesis, OCs become giant and multinucleated through cell-cell fusion, incomplete cytokinesis, and a nuclear recycling process described as fission and fusion18,19,20. The ability to differentiate OCs in vitro has allowed for significant advances in the understanding of bone biology21. OCs differentiate from precursors upon exposure to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). The latter is essential for the normal development and function of OCs in vitro and in vivo, even under inflammatory conditions6,22,23. RANKL is presented by osteoblasts and osteocytes, as well as by activated T cells and fibroblasts in the inflamed RA synovium2,24,25. During the OC differentiation process, monocytes exposed to M-CSF upregulate receptor activator of nuclear factor kappa-B (RANK) expression on their cell membrane and, under subsequent stimulation with RANKL, differentiate into tartrate-resistant acid phosphatase (TRAP)-positive mononuclear pre-OCs and then into multinucleated OCs15,26. OCs produce several enzymes, the chief among them is TRAP, which enables the degradation of phosphoproteins within bone27. A regulator and marker of OC differentiation is the OC-associated receptor (OSCAR). It is upregulated early in precursor cells committing to the OC lineage28. Mature giant multinucleated OCs can degrade (resorb) the skeletal matrix by generating a large sealing zone, which is made of an actin ring surrounding a ruffled border21,29,30. The bone resorption capability of OCs requires cytoskeleton reorganization and the consequent polarization and formation of a convoluted membrane, which is the so-called ruffled border. The ruffled border is surrounded by a large circular band of an F-actin-rich structure, which is the actin ring or sealing zone. Actin ring integrity is essential for OCs to resorb bone both in vitro and in vivo, and defective ruffled border formation is associated with lower vacuolar adenosine triphosphatase (V-ATPase) expression31,32,33. Moreover, OCs are mitochondria-rich cells, and adenosine triphosphate (ATP) associates with mitochondrial-like structures in OCs localized at the ruffled border31,32,33. Rotenone acts as a strong inhibitor of the mitochondrial complex I and impacts ATP production. Rotenone has also been shown to inhibit OC differentiation and function34.
This protocol describes an efficient and optimized method of in vitro osteoclastogenesis&#160;from human peripheral blood samples. In human peripheral blood, CD14+ monocytes are the main source of OCs15,35,36. In this protocol, the kinetics of exposure and the concentrations of M-CSF and RANKL have been adjusted for optimum osteoclastogenesis. Mononuclear cells are first separated from the erythrocytes and granulocytes present in whole blood by&#160;density gradient; they are then enriched for CD14+ monocytes using positive selection by magnetic beads. The isolated CD14+ monocytes are then incubated overnight with M-CSF. This primes the monocytes to upregulate the expression of RANK15,26. The subsequent addition of RANKL induces osteoclastogenesis and multinucleation in a time-dependent manner. Active-resorbing OCs show the characteristic distribution of F-actin rings at the edge of the cell membrane30,32 and staining for TRAP. Mature OCs are analyzed by quantifying TRAP+ multinucleated (more than three nuclei) cells. The functional capacity of mature OCs can be assessed by their resorption, actin ring integrity, and ATP production. Furthermore, differentiated CD14&#8722; OSCAR+ OCs can be enriched and used to assess the effects of certain compounds on OC functionality via mineral (or dentine) resorption and F-actin organization. Additionally, in this work, a known OC inhibitor, rotenone, is used as an exemplar of a compound that affects the functionality of OCs. Reduced OC resorption activity under rotenone is associated with reduced ATP production and actin ring fragmentation. In conclusion, this protocol establishes a robust assay that can be used as a reference method to study several biological aspects of OC differentiation and function in vitro. 
This methodology can be used to assess (1) the potential of circulating monocytes to differentiate into OCs in health and diseases, as well as (2) the impact of therapeutic candidates on OC differentiation and function. This robust osteoclastogenesis protocol enables the determination of the efficacy and mechanisms of bone-targeted therapies on both OC differentiation from precursor cells and the function of mature OCs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide 18 well chamber slides</td>
			<td>ibidi</td>
			<td>81816</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well glass chamber slides&#160;</td>
			<td>Ibidi&#160;</td>
			<td>80807&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well TC plate&#160;</td>
			<td>Corning&#160;</td>
			<td>3596&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well osteo assay stripwell plate&#160;&#160;</td>
			<td>Corning&#160;</td>
			<td>3989&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate solution</td>
			<td>Sigma Aldrich</td>
			<td>386-3</td>
			<td>from kit Cat No. 387A-1KT</td>
		</tr>
		<tr>
			<td>Acetone&#160;</td>
			<td>VWR&#160;</td>
			<td>20066.330&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid phosphatase, Leukocyte (TRAP) kit&#160;</td>
			<td>SIGMA-ALDRICH&#160;</td>
			<td>387A-1KT&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Theremo Fisher - Invitrogen&#160;</td>
			<td>A12379&#160;</td>
			<td>AF488&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Phalloidin</td>
			<td>Thermo Fisher - Invitrogen</td>
			<td>A22287</td>
			<td>AF647</td>
		</tr>
		<tr>
			<td>Alfa Aesar 2-Deoxy-D-glucose&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11321867&#160;</td>
			<td>2DG, 98%&#160;</td>
		</tr>
		<tr>
			<td>Alpha minimum essential medium&#160;</td>
			<td>gibco&#160;</td>
			<td>22571-020&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ATPlite 1step&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>6016731&#160;</td>
			<td>Luminiscence ATP detection assay system&#160;</td>
		</tr>
		<tr>
			<td>BD FACSAria III cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9418-100G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture microplate, 96-well, PS, F-bottom&#160;</td>
			<td>Greiner bio-one&#160;</td>
			<td>655083&#160;</td>
			<td>White-bottom plates&#160;</td>
		</tr>
		<tr>
			<td>Citrate solution</td>
			<td>Sigma Aldrich</td>
			<td>91-5</td>
			<td>from kit Cat No. 387A-1KT</td>
		</tr>
		<tr>
			<td>Corning 6ml round-bottom polystyrene test tubes</td>
			<td>Fisher Scientific</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning osteo assay surface multiple well plate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>CLS3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning osteo assay Surface multiple well plate 1 x 8 stripwell</td>
			<td>Corning</td>
			<td>CLS3989-2EA</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Theremo Fisher &#160;</td>
			<td>D3571&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep human CD14 positive selection kit&#160;</td>
			<td>STEMCELL Technologies&#160;</td>
			<td>17858&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep red blood cell lysis buffer (10x)</td>
			<td>StemCell Technologies&#160;</td>
			<td>20110</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience fixable viability dye eFluor 780</td>
			<td>Theremo Fisher - Invitrogen&#160;</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E7889-100ML&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL auto imaging system</td>
			<td>Thermo Fisher</td>
			<td>A32678</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon round-bottom polypropylene test tubes with cap</td>
			<td>Fisher Scientific</td>
			<td>10314791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 mL</td>
			<td>Corning&#160;</td>
			<td>430790&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 mL&#160;</td>
			<td>Corning&#160;&#160;</td>
			<td>430828&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Garnet GBC base solution</td>
			<td>Sigma Aldrich</td>
			<td>387-2</td>
			<td>from kit Cat No. 387A-1KT</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>gibco&#160;</td>
			<td>10500-064&#160;</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>cytiva&#160;</td>
			<td>17144003&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F-8775&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Human sRANK ligand&#160;</td>
			<td>PEPROTECH&#160;</td>
			<td>310-01-100UG&#160;</td>
			<td>Receptor activator of nuclear factor kappa-B ligand&#160;(RANKL)</td>
		</tr>
		<tr>
			<td>ImageJ Image analysis software</td>
			<td>Image J</td>
			<td>version 2.9.0</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</td>
			<td>gibco&#160;</td>
			<td>25030-024&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium heparin tubes (9 mL)&#160;</td>
			<td>VACUETTE&#160;</td>
			<td>455084&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony-stimulating factor&#160;</td>
			<td>PEPROTECH&#160;</td>
			<td>300-25-100UG&#160;</td>
			<td>&#160;M-CSF</td>
		</tr>
		<tr>
			<td>Napthol AS-BI phosphoric acid solution</td>
			<td>Sigma Aldrich</td>
			<td>387-1</td>
			<td>from kit Cat No. 387A-1KT</td>
		</tr>
		<tr>
			<td>Neubauer hemacytometer counting chamber&#160;</td>
			<td>Camlab</td>
			<td>SKU 1127885</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin from Streptomyces Diastatochromogenes&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Q4876-5MG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>OSCAR Antibody, anti-human, Vio Bright FITC, REAfinit</td>
			<td>Miltenyi Biotec</td>
			<td>130-107-661 and 130-107-617</td>
			<td>Clone REA494</td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-human CD14 antibody</td>
			<td>Biolegend</td>
			<td>325618</td>
			<td>Clone HCD14&#160;</td>
		</tr>
		<tr>
			<td>Penicilin/streptomycin&#160;</td>
			<td>SIGMA&#160;</td>
			<td>P0781&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PHERAstar machine and software</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (DPBS, 1x)&#160;</td>
			<td>gibco&#160;</td>
			<td>14190-094&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>REA control antibody (S), human IgG1, Vio Bright FITC, REAfinity</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-443</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>425044-1L&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite solution</td>
			<td>Sigma Aldrich</td>
			<td>91-4</td>
			<td>from kit Cat No. 387A-1KT</td>
		</tr>
		<tr>
			<td>Tartrate solution</td>
			<td>Sigma Aldrich</td>
			<td>387-3</td>
			<td>from kit Cat No. 387A-1KT</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>9002-93-1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T8154-100ML&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation of functional osteoclasts from human peripheral blood cd14+ monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

OC generation from CD14+ monocytes 
This method aimed to easily differentiate a large number of OCs from human peripheral blood CD14+ monocytes in vitro, typically in 1 week. Firstly, CD14+ monocytes were enriched from PBMCs and primed with M-CSF overnight to upregulate RANK, as previously reported15. Following monocyte priming, to determine the optimum concentration of RANKL for OC differentiation and maturation, RANKL concentrations of 1 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL, along with 25ng/mL M-CSF, were used. The addition of RANKL produced increasing numbers of large TRAP-positive multinucleated OCs in a dose-dependent manner, and this was assessed using TRAP staining. Mature OCs are defined as TRAP-positive cells with multiple nuclei (typically more than three; Figure 2A,B and Supplementary Figure 1). Furthermore, the kinetics of OC differentiation from monocytes were investigated using TRAP staining and light microscopy over a 2-14 day culture period. In this instance, OC differentiation using an intermediate concentration of 50 ng/mL RANKL was chosen to assess how fast OCs differentiated in culture. In these culture conditions, multinucleated OCs were visible from day 5 onward, and optimal differentiation was reached on day 7 (Figure 2C). The prolonged incubation of cultures beyond 10 days on plastics resulted in abnormally giant fused cells. In this protocol, days 6-8 are usually used as the optimal endpoint of OC generation. The OCs can be quantified or used for downstream assays.
Functional assessment of differentiated OCs 
To determine the functional activity of the generated OCs, we examined their resorptive activity by differentiating the OCs on a mineralized surface. As large OCs are only generated after a 7 day culture period, and to allow sufficient time to resorb the mineral substrate, the cultures were maintained until day 10. The formation of round holes, or resorption pits, was observed only on the mineralized surfaces of wells containing cells that had been treated with both M-CSF and RANKL (Figure 3). Thus, the percentage of dissolved mineralized surface (resorption pits) allows for determining the OC resorptive capacity. Additionally, the OCs differentiated following this protocol up to day 7, both on plastic and glass chamber slides, displayed a well-organized actin ring structure that could be visualized by immunofluorescent staining (Supplementary Figure 2).
Effect of an inhibitor on mature OC function 
The above mentioned culturing conditions were utilized to determine the functional capability of the in vitro generated OCs&#160;in the presence of the known OC inhibitor, rotenone34. The OCs were differentiated for 6-8 days, and CD14&#8722;OSCAR+ OCs and OC precursors were enriched via flow cytometry (Figure 4). The enriched cells were then plated at 50,000 cells/per well onto a mineral-coated 96-well plate in pro-osteoclastogenic medium (25 ng/mL M-CSF and RANKL) for 3 days. Treatment with rotenone (Figure 5A,B) dose-dependently inhibited the resorption of the mineralized surface in comparison with the untreated control well, consistent with previous studies34. Additionally, OC functionality was assessed via ATP production and actin ring formation. The rotenone-dependent inhibition of OC resorption was associated with the inhibition of ATP production (Figure 5C). Resorbing OCs are highly polarized cells that regulate their resorptive capacity by promoting cytoskeletal organization. Alexa fluor 647 conjugated phalloidin was used to label the F-actin cytoskeleton of the mature OCs cultured in the presence or absence of rotenone. Rotenone caused the fragmentation of the RANKL-derived actin ring of the mature OCs (Figure 5D).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64698/64698fig02.jpg" /> 
Figure 2: OCs efficiently differentiating from CD14+ monocyte precursors. CD14+ monocytes were magnetically enriched, plated at 1 x 105 cells/well in 96-well plates, and incubated overnight with 25 ng/mL M-CSF. (A) M-CSF-primed monocytes were stimulated with increasing concentrations of RANKL (1 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL), fixed, and stained for TRAP on day 7. Images were acquired, and the TRAP+ multinucleated cells (MNCs) were counted. Representative images of TRAP staining are shown in Supplementary Figure 1. The error bars show mean &#177; SD (n = 3). The data were analyzed with a one-way ANOVA and Holm-Sidak&#39;s multiple comparisons test for paired data; * P &#8804; 0.05 and ** P &#8804; 0.005.&#160;(B) Representative image of a TRAP-stained well of a 96-well plate showing the typical amount of OCs/well expected and their morphology under 25 ng/mL RANK-L in comparison to M-CSF-derived macrophages at day 7. Scale bars: 1000 &#181;m. (C) Representative images of OC formation under 50 ng/mL RANKL assessed via TRAP staining from day 2 to day 14. OCs are visible from day 5 onward. Giant abnormally fused OCs are present after 10 days. Scale bars: 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/64698fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64698/64698fig03.jpg" /> 
Figure 3: Resorptive OCs differentiated from CD14+ monocytes. CD14+ cells isolated from PBMCs were differentiated for 10 days into OCs in the presence of 25 ng/mL M-CSF (M) and RANKL (R) on mineral assay surface (osteo-assay) plates. (A) Images of representative reconstructed wells taken at 10x magnification to analyze the resorption on day 10 (mineral substrate in gray; resorption pits in white). Scale bars: 1000 &#181;m. (B) Quantification of the percentage of resorbed area. The resorption data were analyzed with a Wilcoxon paired analysis. The error bars show mean &#177; SD (n = 7). <a href="https://www.jove.com/files/ftp_upload/64698/64698fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64698/64698fig04.jpg" /> 
Figure 4: Flow cytometry enrichment of CD14&#8722;OSCAR+ OCs. CD14+ monocytes were enriched from PBMCs, and the OCs were differentiated as previously described. Adherent OC cultures were detached with accutase and stained for flow cytometry. (A-C) OCs at day 8 were sorted based on CD14 and OSCAR expression. (A) Representative sorting gating strategy. The cells were gated as singlets, negative for dead staining, and the CD14+ OSCAR+ (red) and CD14&#8722; OSCAR+ (blue) subsets were sorted. (B) Representative plots showing the overlapping OSCAR staining of RANKL-derived OCs (cyan) and control M-CSF-derived macrophages (orange). In red is the OSCAR isotype-stained control of RANKL-derived OCs. (C) The sorted populations were plated on plastic and allowed to adhere for 2 h in pro-OC medium (25 ng/mL M-CSF and 50 ng/mL RANKL), followed by TRAP staining and visualization. The representative images show a lack of TRAP+ cells in the CD14+ subset (red) and mono- and multinucleated TRAP+ pre-OCs and OCs in the CD14&#8722; subset (blue). Scale bars: 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/64698fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64698/64698fig05.jpg" /> 
Figure 5: Assays to assess the function of mature OCs. To evaluate the function of mature OCs, CD14+ cells isolated from PBMCs were cultured either with M-CSF (M) alone or combined with RANKL (R) for 7 days, the OCs were enriched via flow cytometry, and the OCs were then treated with the inhibitor rotenone for 24 h. (A) Mature OCs were sorted via flow cytometry (CD14&#8722;OSCAR+) and were cultured on a mineral assay surface in the presence or absence of rotenone for 3 days, after which the cells were bleached and imaged at 10x to reveal the resorbed area (resorption pits in white). (A) Representative reconstructed images of wells. Scale bars: 1000 &#181;m. (B) The quantification of the percentage of resorbed area. The data in (B) were analyzed with a one-way ANOVA with Dunn&#39;s multiple comparisons test (n = 7); * P &#8804; 0.05 and** P &#8804; 0.01. The error bars show the mean &#177; SD. (C) Total intracellular ATP content of undifferentiated and day 7 differentiated mature OCs differentiated with RANKL and treated with either vehicle or rotenone (10 nM and 30 nM). Here, 2DG and oligomycin were used as positive controls for the assay and were added 30 min prior to cell lysis and ATP quantification. The error bars show the mean &#177; SD (n = 4). The data were analyzed with a one-way ANOVA and Dunnett&#39;s multiple comparison test for paired data. ** P &#8804; 0.01. (D) A representative 20x image of mature OCs stained for actin ring formation (red) and nuclei (blue), showing the loss of the actin ring with the inhibitor. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/64698fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plate format</td>
			<td>96 well-plate</td>
			<td>48 well-plate</td>
			<td>24 well-plate</td>
			<td>12 well-plate</td>
			<td>6 well-plate</td>
		</tr>
		<tr>
			<td>volume</td>
			<td>100 &#181;L</td>
			<td>225&#8211;250 &#181;L</td>
			<td>450&#8211;500 &#181;L</td>
			<td>0.8&#8211;1 mL</td>
			<td>1.8&#8211;2 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Volume of cell suspension for different plate formats. The volumes are calculated starting from a 1 x 106 cells/mL solution and provide an optimum density for cell-cell fusion.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Fluorophore, clone&#160;&#160;</td>
			<td>Volume (&#956;L) per 106 cells</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>PE/Cyanine7, HCD14&#160;</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td>OSCAR</td>
			<td>FITC, REA494</td>
			<td>10 &#956;L&#160;</td>
		</tr>
		<tr>
			<td>Cell sorting buffer&#160;</td>
			<td></td>
			<td>80 &#956;L&#160;</td>
		</tr>
	</tbody>
</table>
Table 2 : Antibody master mix solution. 
Supplementary Figure 1: TRAP staining of the RANKL dose response. CD14+ monocytes were magnetically enriched, plated at 1 x 105 cells/well in 96-well plates, and incubated overnight with 25 ng/mL M-CSF, as in Figure 2. Representative images of TRAP staining show MCSF-primed monocytes stimulated with increasing concentrations of RANKL (1 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL), fixed, and stained for TRAP on day 7. Scale bars: 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/Supl. Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Acting ring staining in fully differentiated OCs. (A) A 10x magnification of OCs differentiated on TC plastic and stained with AF647 phalloidin (in red). Scale bar: 400 &#181;m. (B) A 40x magnification of OCs differentiated on glass chamber slides and stained with AF488 phalloidin (in yellow). Scale bar: 100 &#181;m.The nuclei are stained with DAPI, shown in blue in (A) and in cyan in (B). <a href="https://www.jove.com/files/ftp_upload/64698/Supl.Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Effect of different FBS batches on OC differentiation efficiency. OCs were differentiated from CD14+ monocytes in the presence of 25 ng/mL M-CSF and 50 ng/mL RANKL (MR) for 7 days. The control wells had M-CSF only (M). (A) Representative 10x magnifications (scale bars: 400 &#181;m) and (B) quantification of TRAP-stained OCs differentiated from one donor in two different batches of FBS. The error bars show the mean &#177; SD of three technical replicates. <a href="https://www.jove.com/files/ftp_upload/64698/suppl.Figure 3.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes at JoVE.com

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

differentiation-functional-osteoclasts-from-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

2.6K Views.  University of Glasgow. Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Video: Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

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 ...

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  &amp;#947;&amp;#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 ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...