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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from naïve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Abstract

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence naïve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from naïve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.

Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

Introduction

Dendritic cells (DCs) are critical mediators between innate and adaptive immunity. DCs, which mainly reside in mucosal membranes, skin and lymphoid tissue, are the primary antigen presenting cells (APCs)1. DCs uptake foreign proteins and process and present them on major histocompatibility (MHC) proteins to naïve T cells. DCs specifically express MHC class II proteins, such as human leukocyte antigen-DR (HLA-DR) in humans. The activation state of the DCs upon antigen exposure is critical for the downstream T cell response2. Immature DCs express various pattern recognition receptors (PRRs) that recognize classes of molecules call pathogen associated molecular patterns (PAMPs), such as the bacterial wall component, lipopolysaccharide (LPS)3. Upon PRR stimulation, DCs become matured DCs and upregulate important T cell co-stimulatory proteins, such as CD80, CD86, and CD40, and secrete pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), facilitating the differentiation of naïve T cells into conventional effector or helper T cells2. On the contrary, if DC maturation is interrupted or if DCs develop in a tolerogenic environment, DCs can generate a tolerogenic DC state (tolDCs)4. TolDCs downregulate classical T cell co-stimulatory receptors and instead upregulate tolerance receptors such as programmed cell death ligand 1 (PD-L1) and B- and T-lymphocyte attenuator (BTLA) and generate suppressive cytokines such as interleukin 10 (IL-10) and transforming growth factor beta (TGF-β)4. This is not a comprehensive list of tolerance markers and, in fact, there is limited consensus as to which tolDC markers are appropriate to define the tolDC state5. Considering this, we propose regulatory T cell (Treg) generation as a functional marker that ought to be used to compare the effectiveness of various tolDC induction agents.

In addition to tolDC/matured DC activation states, DCs can also be categorized based on their lineage or tissue location, with each subset displaying slightly different functionality. While the tolDC/matured DC division is less definitive and exists more as a continuum, lineage divisions have well-defined markers in both human and mice. DC precursors are formed in the bone marrow, but there are two main subtypes of DCs based on their lineage: 1) plasmacytoid dendritic cell (pDCs), which derive from lymphoid lineages and 2) conventional dendritic cells (cDCs), which derive from myeloid lineages. In humans, pDCs are matured in lymphoid organs, express CD303, and are highly responsive in viral infections6. CD11c expressing cDCs, meanwhile, are matured in peripheral tissues and exist in two distinct subtypes, CD1c+ cDC1s and CD141+ cDC2, which each generate distinct T cell responses7. Furthermore, all cDCs can exist in either tissue-resident (CD103-) or migratory (CD103+) substates8. Finally, under certain conditions, cells from monocyte lineages (CD14+) can be induced toward a dendritic cell phenotype and are identified as CD14-, CD141+, CD1c+ 9. These cells, known as monocyte-derived DCs (moDCs), are the most commonly used for ex vivo analysis in humans as monocytes constitute approximately 10-30% of human peripheral blood mononuclear cells (PBMCs), whereas pDCs constitute only 1-3%10. This makes moDCs an attractive choice, but it is also known that moDCs are more inflammatory that typical cDCs isolated from primary tissue9.

There are currently two broad categories of efforts to employ tolDCs to generate clinical tolerance. First, tolDCs are generated from monocytes for use as a cell therapy. In this paradigm, moDCs are typically differentiated using IL-4/GM-CSF concurrently with immunomodulators such as vitamin D3, rapamycin (rapa), IL-10, dexamethasone, or combinations of these11,12. These tolDCs have been explored as autologous cell therapies for autoimmunity and transplants13. The other use of tolDCs is to reprogram endogenous DCs towards tolDCs using free drugs or nanocarriers to deliver both immunomodulator and antigen of interest14,15,16. Induction of already differentiated DCs is more challenging, however, due to the development of robust metabolic phenotypes of DCs that are typically in contrast with tolDC metabolism17,18. This is a high bar for most pharmacological immunomodulators; for this reason, most endogenous DC reprogramming studies report effective DC suppression and often some Treg induction, but lack clinical success, often due to lack of T cell persistence15,19,20. This highlights the need for strategies to identify potential tolDC induction agents from existing DCs.

Here, we present a method for in vitro evaluation of immunomodulatory agents against differentiated moDCs with the end metric of autologous Treg induction. This protocol is designed to assess the effectiveness of immunomodulatory agents to reprogram already-differentiated human moDCs towards tolerance. Furthermore, this protocol validates the functionality of reprogrammed tolDCs to generate Tregs against autologous T cells isolated from the same PBMC sample. This is in contrast to other protocols that induce tolerance during differentiation and/or challenge tolDCs with T cells from allogenic donors21. In this protocol, we use the common tolerizing agent rapa as an example but also demonstrate the limited effectiveness of rapa-treated moDCs to generate Tregs. In our representative results, we also show the efficacy of other common immunomodulatory treatments such as IL-10, dexamethasone, and vitamin D3. We envision this protocol being used to screen potentially more effective tolDC-inducting agents against already established moDCs22.

Protocol

All human peripheral blood mononuclear cell (PBMC) samples were obtained from the University of Pennsylvania's Human Immunology Core from deidentified donors with prior approval from the University of Pennsylvania's Institutional Review Board (IRB) with patient consent.

Optional: While in this method, we used freshly isolated PBMCs obtained from an academic laboratory, PBMCs can be isolated from either whole blood or leukapheresis-enriched blood products. We recommend using the density gradient centrifugation method, as this is a well-established and reliable method that is described elsewhere23.

1. Isolation of monocytes/T cells and moDC differentiation

  1. Isolation of monocytes and T cells from PBMCs-Day 1
    1. Obtain 200 million human PBMCs from a healthy donor. Keep the cells in a 15 mL tube and transfer them on ice.
    2. Aliquot 50 mL of Separation Buffer (provided in commercially available separation kits) in a conical tube.
      NOTE: Separation buffer can also be replaced with DPBS with 2% fetal bovine serum (FBS) and 1 mM Ethylenediaminetetraacetic acid (EDTA).
    3. Remove the PBMC tube from ice and fill it with Separation Buffer. Centrifuge at 300 x g for 5 minutes.
    4. Aspirate the supernatant using a 10 mL serological pipette. Resuspend the cell pellet in 4 mL of Recommended Medium using a serological pipette.
    5. Divide the suspension into two different 15 mL tubes, adding 2 mL per tube (5 × 107 cells/mL per tube). Label one tube "T" and the other "Monocytes."
    6. Retrieve the Human Monocyte Isolation Kit and follow the manufacturer's protocol to isolate monocytes from the monocyte-labeled tube.
    7. Retrieve the Human T Cell Isolation Kit and follow the manufacturer's protocol for the second tube to isolate T cells.
  2. Plating of monocytes for differentiation and freezing of T cells-Day 1
    1. Preheat 50 mL of moDC Culture Medium (Table 1) at 37 °C for at least 10 min.
    2. Centrifuge the 15 mL tubes with enriched monocytes and the 15 mL tube with T cells from step 1.1 at 250 × g for 5 min.
    3. Aspirate the supernatant. Resuspend monocytes in 1 mL of moDC Culture Medium and T cells in T Cell Culture Medium (Table 1) by pipetting up and down. Ensure the pellet is properly dispersed.
    4. Count the cells using an automated cell counter or manual hemacytometer using Trypan Blue staining.
      NOTE: For the representative data, 10 million T cells and 3 million monocytes were isolated.
    5. Take the tube with monocytes and add 9 mL of warm moDC Culture Medium to the suspension for a final volume of 10 mL. Then, add 100 µL each of 10 µg/mL GM-CSF and 10 µg/mL IL-4 stock for a final concentration of 100 ng/mL of both GM-CSF and IL-4 in the media. Transfer the suspension to a Petri dish and label as "moDC" (Day 1) and incubate at 37 °C with 5% CO2.
    6. Take the T cell tube and mix 1 mL of T Cell Freeze Medium (Table 1) with 1 mL of T cell suspension. Divide into two 2 mL cryovials (1 mL/vial); seal tightly (final concentration of 5 million cells per vial). Place the cryovials in a freezing chamber with balancing tubes in unused wells. Store the chamber at −80 °C overnight, then transfer the cryovials to a cryotank.
    7. On Day 4, refresh the medium with the addition of 5 mL of fresh moDC Culture Medium and add 100 µL each of GM-CSF and IL-4 stock. Differentiated moDCs will be ready on Day 7.

2. Adding immunomodulatory drugs for generating tolerogenic moDCs

  1. Set up the moDC plate.
    1. On Day 7, retrieve the differentiated moDCs from the incubator and transfer the moDC cell suspension into a 50 mL tube.
    2. Centrifuge the suspension at 250 × g for 5 min. Aspirate the supernatant and resuspend the cell pellet in 1 mL of warm moDC culture media by pipetting up and down. Count the cells with hemacytometer and dilute the cells to a concentration of 3 × 105 cells/mL in warm moDC culture medium containing 100 ng/mL GM-CSF and IL-4.
    3. Add 3 × 104 cells/well in 100 µL to the desired number of wells in a flat bottom 96-well tissue culture plate.
      NOTE: For representative results, we used a total of 48 wells (four conditions done in triplicate prepared for four different analyses in sections 3 and 4), but this can be adjusted for up to 60 wells. Fill all remaining wells with 100 µL/well PBS to prevent drying, especially outer wells. We recommend preparing different plates for each method of analysis.
    4. Add 1 µL/well of immunomodulatory drugs to the desired wells (here 10 ng/mL rapa). Incubate the plate overnight at 37 °C with 5% CO2.
      NOTE: If including immunostimulation on day 8, prepare wells for the immunomodulatory drug both with and without immunostimulation. Use of immunostimulation to mature moDCs is optional.
  2. Rethaw T cells.
    1. On Day 8, retrieve two cryovials from the cryotank and thaw the cryovials in a hot bead bath or water bath at 37 °C until the contents start melting (in <3 min).
    2. As the contents start to melt, transfer the vials to a cell culture hood and pipet 1 mL of warm T cell culture media into the vial to rapidly thaw. Transfer the entire contents of the vial into a 50 mL tube. Rinse the cryovials with 1 mL of T cell culture medium to ensure all cells are transferred into the tube.
    3. Top up the suspension with Flow Staining Solution (Table 1) to 15 mL. Centrifuge at 200 × g for 10 min and aspirate the supernatant. Then, resuspend the pellet in 5 mL of Flow Staining Solution and centrifuge again.
    4. Aspirate the supernatant and resuspend the cells in 1 mL of warm T cell culture medium. Count the cells using a hemacytometer.
      NOTE: Viability should be >70% at this stage; if large amounts of dead cells remain, transfer to a fresh tube, add 10 mL of cell stain solution, centrifuge at 200 × g for 5 min, resuspend in 1 mL of T cell media, and recount.
    5. Add 9 mL of warm T cell culture medium to make a final volume of 10 mL. Transfer the suspension into a Petri dish. Label it as "Rethawed T Cells" and incubate overnight at 37 °C with 5% CO2 to let the cells rest.
  3. Wash the moDC plate and add the immunostimulator if desired.
    1. On Day 8, centrifuge the moDC plate (from Day 7) at 300 × g for 5 min.
    2. Aspirate the supernatant, wash with warm HBSS, repeat centrifugation, aspirate again, and resuspend the cells in warm moDC culture medium containing GM-CSF and IL-4 (100 µL/well).
      NOTE: Be careful not to disturb the cells. Tilt the plate during aspiration. Approximately 10 µL can remain in the well.
    3. If using immunostimulation, add 1 µL/well of 100x stock LPS (or other immunostimulatory agent) to the desired wells. Incubate the plate overnight at 37 °C with 5% CO2.

3. Flow analysis for moDC (Validation + Tolerance)

  1. Flow analysis for validation of moDC
    1. On day 8, prepare antibody cocktails for the Validation Panel, targeting the following markers: HLA-DR, CD14, dendritic cell-specific C-type lectin (DC-SIGN), CD1c, and CD40 as described in Table 2. Dilute all antibodies with flow stain solution.
      NOTE: The panel was designed for a 7 channel, Red/Blue laser flow cytometer.
    2. Retrieve the moDC plate from the incubator. Centrifuge the plate at 300 × g for 5 min. Remove the supernatants. Optional: Retain the supernatants for ELISA analysis for cytokines IL-10 and TNFα using commercially available kits according to the manufacturers' instructions.
    3. Add 200 µL/well of Flow Staining Solution. Centrifuge the plate at 300 × g for 5 min.
    4. Aspirate the supernatant and add 50 µL/well of Fc receptor binding inhibitor antibody diluted 1:200 in flow staining solution to block unspecific binding. Incubate at room temperature (RT) for 30 min.
    5. Add 50 µL/well of the prepared Validation Panel antibody cocktail.
    6. Prepare compensation controls in the plate or in 1.5 mL tubes by mixing 50 µL of compensation beads with 50 µL of each diluted antibody.Incubate the plate at 4 °C for 1 h.
      NOTE: Compensation beads are used here rather than cells for compensation due to potentially low expression of markers on moDCs.
    7. Wash by centrifuging at 300 × g for 5 min, removing the supernatant, and resuspending in 200 µL of Flow Staining Solution. Perform two washes.
    8. After the final wash, centrifuge again and resuspend the cells in 110 µL/well of Flow Staining Solution. The samples are now ready for flow cytometry analysis.
  2. Flow analysis for tolerogenic moDC (Tolerance Panel)
    1. Similar to step 3.1 on day 8, prepare antibody cocktails for the Tolerance Panel, targeting the following markers: CD86, PD-L1, DC-SIGN, CD1c, BTLA, and CD40 (Table 2).
    2. Repeat steps 3.1.2-3.1.8, substituting the antibody cocktail with the Tolerance Panel cocktail.

4. Flow analysis of T cells

  1. Combine T cells with treated Tolerogenic moDCs.
    1. On day 9, retrieve the rethawed T cell Petri dish from the incubator and transfer all T cells into a 50 mL tube. Top up with Flow Staining Solution.
    2. Optional: If performing T cell proliferation analysis, divide T cells into two tubes.
      1. With the first tube, spin down at 250 × g for 5 min and remove the supernatant. Stain this tube with cell proliferation dye according to the manufacturer's instructions.
        NOTE: We used Fixable Viability Dye eFluor 670, which requires a 30 min incubation step, followed by two washing steps. We suggest using 50 mL conical tubes to prevent cell loss and using HBSS with 10% HIFBS to wash after staining.
      2. Use the second tube containingunstained T cells for Treg analysis. Similarly spin down at 250 × g for 5 min, remove the supernatant, resuspend in warm cell culture media, and keep in the incubator during the staining process. If no T cell proliferation is required, spin down all T cells and replace them with warm T cell culture media.
    3. Count the cells and obtain at least 4 million (for just Treg analysis) or 8 million (for Treg and T proliferation analysis).
    4. Retrieve the moDC plate from incubator with the remaining wells, centrifuge at 300 × g for 5 min, and apirate the supernatants. Add 200 µL/well T cells at 1.6 × 105 cells/well. Add unstained T cells for Treg analysis plates and stained T cells for T cell proliferation plates.
      NOTE: This will maintain a T cell to moDC ratio of 5:1, as the initial 3 ×104 moDCs typically expand slightly.
    5. Add 25 µL/mL of anti-CD3/CD28 antibody cocktail to all groups except negative control wells to stimulate T cells. Incubate the plate for 72 h at 37 °C with 5% CO2 until day 12.
      NOTE: Be sure to use stained T cells for proliferation analysis and unstained for Treg analysis.
  2. Treg flow analysis
    1. Surface marker staining
      1. On day 12, prepare antibody cocktails for the Treg Panel (Table 2).
      2. Transfer all cell suspensions from the 96-well culture plate into a V-bottom plate with Flow Staining Solution. Retain the supernatants for cytokine analysis.
      3. Wash the cells 2x by centrifugation (200 × g for 5 min followed by 200 µL of Flow stain solution). Set aside some cells for compensation controls. Set one aside for FOXP3 staining and for unstained controls.
        NOTE: We suggest combing 10 µL of all samples prior to the final spin in step 4.2.1.3 to obtain necessary compensation controls. This provides a representative sample of all surface level expression of each marker on the cells.
      4. After the second wash, centrifuge at 200 x g for 5 min at room temperature and aspirate, then add 50 µL/well Fc receptor binding inhibitor antibody (diluted 1:200 in Flow Staining Solution). Incubate at room temperature for 10 min.
      5. Add 50 µL/well of the prepared Treg Panel antibody cocktail.
      6. For compensation controls, mix 50 µL of unstained compensation cells with 50 µL of each single stained antibody.
      7. Incubate the plate and compensation controls at 4 °C for 1 h. Wash the plate once with Flow Stain Solution and centrifuge at 300 × g for 5 min.
      8. Stain with Live/Dead Near-IR dye diluted in HBSS (1:1,000) at 200 µL/well. Incubate at 4 °C for 30 min.
      9. Wash the plate once with Flow Staining Solution (200 µL/well) at 300 × g for 5 min.
    2. Stain with FoxP3 according to the manufacturer's instructions, which will require an overnight incubation. A brief overview of the FoxP3 staining protocol is described below .
      1. Prepare the FoxP3 Fixation/Permeabilization Working Solution according to the manufacturer's instructions. Mix 1 part of FoxP3 Fixation/Permeabilization Concentrate with 3 parts of FoxP3 Fixation/Permeabilization Diluent. Prepare 200 µL of the working solution per sample.
      2. Incubate all samples in 200 µL/well of the working Fixation/Permeabilization solution. Leave at 4 °C for 1 h.
      3. Prepare 1x Permeabilization Buffer: Mix 1 part of 10x Permeabilization Buffer (obtained from commercial kit) with 9 parts of distilled water.
      4. Wash the plate 2x with 1x Permeabilization Buffer (200 µL/well) at 300 × g for 5 min.
      5. Stain with FoxP3 antibody (PE-Cy5.5, 1:300 dilution in 1x Permeabilization Buffer): Add 100 µL/well of the diluted FoxP3 antibody and incubate at 4 °C overnight.
      6. Wash the plate 2x with 1x Permeabilization Buffer (200 µL/well) at 300 × g for 5 min.
      7. Resuspend the cells in 110 µL/well of Flow Staining Solution.
      8. Perform flow cytometry analysis.
  3. T cell proliferation analysis
    1. Prepare antibody cocktails for the T Proliferation Panel (Table 2).
    2. Transfer all cell suspensions from the 96-well culture plate into a V-bottom plate with Flow Staining Solution. Retain supernatants for cytokine analysis.
    3. Wash the cells 2x by centrifugation (200 × g for 5 min followed by 200 µL of Flow stain solution). Set aside some cells for compensation controls. Set one aside for FOXP3 staining and for unstained controls.
      NOTE: We suggest combing 10 µL of all samples prior to the final spin in step 4.3.3 to obtain the necessary compensation controls. This provides a representative sample of all surface level expression of each marker on the cells.
    4. After the second wash, centrifuge (200 × g for 5 min at room temperature) and aspirate, then add 50 µL/well Fc receptor binding inhibitor antibody (diluted 1:200 in Flow Staining Solution). Incubate at room temperature for 10 min.
    5. Add 50 µL/well of the prepared T Proliferation Panel antibody cocktail.
    6. For compensation controls, mix 50 µL of unstained compensation cells with 50 µL of each single stained antibody.
    7. Incubate the plate and compensation controls at 4 °C for 1 h. Wash the plate once with Flow Stain Solution and centrifuge at 300 × g for 5 min.
    8. Stain with Live/Dead Near-IR dye diluted in HBSS (1:1,000) at 200 µL/well. Incubate at 4 °C for 30 min.
    9. Wash the plate once with Flow Staining Solution (200 µL/well) at 300 × g for 5 min.
    10. Perform flow cytometry analysis.

Results

We have described a protocol for human PBMCs, isolate both CD3+ T cells and CD14+ monocytes using commercially available magnetic separation kits, differentiate monocytes into CD14-, HLA-DR+, CD141+, CD1c+ moDCs using GM-CSF and IL-4, treat them for 24 h, and co-culture with autologous T cells with anti-CD3/CD28 stimulation for 72 h. An experimental schematic is shown in Figure 1.

Isolation ...

Discussion

Here we describe a reliable and versatile method to assess the functionality of immunomodulatory agents to induce tolDCs from moDCs and validate their functionality to generate Tregs from autogenic T cells ex vivo. There are several critical steps in this protocol. First, monocytes are notoriously sensitive cells and must be obtained from fresh, not previously frozen PBMCs for the best results. Monocytes should be isolated as soon as possible and placed in the differentiation cocktail. Typically, poor monocyte y...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

We would like to thank the University of Pennsylvania's Human Immunology Core (HIC) for providing fresh human PBMCs from donors. The HIC is supported in part by NIH P30 AI045008 and P30 CA016520.

Materials

NameCompanyCatalog NumberComments
0.1-10 µL Filtered Pipet tipsVWR76322-158General Cell Culture
1.5 mL Centrifuge TubeVWR77508-358General Cell Culture
10 mL Serological PipetsVWR414004-267General Cell Culture
100-1000 µL Filtered Pipet tipsVWR76322-164General Cell Culture
15 mL Conical TubeVWR77508-212General Cell Culture
20-200 µL Filtered Pipet tipsVWR76322-160General Cell Culture
2-MercaptoethanolMP Biomedical194834T Cell Culture
50 mL Conical TubeVWR21008-736General Cell Culture
60 x 15 mm Dish, Nunclon DeltaThermo Fischer150326General Cell Culture
96 Well Conical (V) Bottom Plate, Non-Treated SurfaceThermo Fischer277143General Cell Culture
96 well Flat Bottom PlateThermo Fischer161093General Cell Culture
APC/Cyanine7 anti-human CD272 (BTLA) AntibodyBiolegend344518Flow Cytometry
Attune NxT (Red/Blue Laser, 7 Channel)Thermo FischerA24863Flow Cytometry
BSAThermo Fischer15260-037General Cell Culture
CD14 Monoclonal Antibody (61D3), PEThermo Fischer12-0149-42Flow Cytometry
CD1c Monoclonal Antibody (L161), PE-Cyanine7Thermo Fischer25-0015-42Flow Cytometry
CD209 (DC-SIGN) Monoclonal Antibody (eB-h209), PerCP-Cyanine5.5Thermo Fischer45-2099-42Flow Cytometry
CD25 Monoclonal Antibody (CD25-4E3), APCThermo Fischer17-0257-42Flow Cytometry
CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH1), PEThermo Fischer12-5983-42Flow Cytometry
CD4 Monoclonal Antibody (RPA-T4), Alexa Fluor 488Thermo Fischer53-0049-42Flow Cytometry
CD40 Monoclonal Antibody (5C3), APCThermo Fischer17-0409-42Flow Cytometry
CD40 Monoclonal Antibody (5C3), APC-eFluor 780Thermo Fischer47-0409-42Flow Cytometry
CD69 Monoclonal Antibody (FN50), PEThermo FischerMA1-10276Flow Cytometry
CD86 Monoclonal Antibody (BU63), FITCThermo FischerMHCD8601Flow Cytometry
CD8a Monoclonal Antibody (RPA-T8), PE-Cyanine7Thermo Fischer25-0088-42Flow Cytometry
Conical Bottom (V-well) 96 Well PlateThermo Fischer2605Flow Cytometry
Cryogenic Vials, 2 mLThermo Fischer430488T Cell Culture
Dimethylsulfoxide (DMSO), Sequencing GradeThermo Fischer20688General Cell Culture
DPBSThermo Fischer14200166General Cell Culture
EasySep Human Monocyte Isolation KitStem Cell Technologies19359Cell Separation
EasySep Human T Cell Isolation KitStem Cell Technologies17951Cell Separation
EasySep MagnetStem Cell Technologies18000Cell Separation
EDTAThermo FischerAIM9260GGeneral Cell Culture
Falcon Round-Bottom Polystyrene Tubes, 5 mLStem Cell Technologies38025Cell Separation
Fc Receptor Binding Inhibitor Polyclonal AntibodyThermo Fischer14-9161-73Flow Cytometry
Fetal Bovine SerumThermo FischerA5670701General Cell Culture
Fixable Viability Dye eFluor 780Thermo Fischer65-0865-18Flow Cytometry
Foxp3 / Transcription Factor Staining Buffer SetThermo Fischer00-5523-00Flow Cytometry
FOXP3 Monoclonal Antibody (PCH101), PE-Cyanine5.5Thermo Fischer35-4776-42Flow Cytometry
HBSSThermo Fischer14170-112General Cell Culture
Heat Inactivated Fetal Bovine SerumThermo FischerA5670801General Cell Culture
HEPES (1 M)Thermo Fischer15630106moDC Cell Culture
HLA-DR Monoclonal Antibody (L243), Alexa Fluor 488Thermo FischerA51009Flow Cytometry
Human CD3/CD28/CD2 T Cell ActivatorStemCell Technologies10970T Cell Culture
Human GM-CSF Recombinant ProteinThermo Fischer300-03moDC Cell Culture
Human IL-10 ELISA Kit, High SensitivityThermo FischerBMS215-2HSELISA
Human IL-4, Animal-Free Recombinant ProteinThermo FischerAF-200-04moDC Cell Culture
Human PBMC (Freshly Isolated)UPenn HICN/ACells
Human TNF alpha ELISA KitThermo FischerBMS223-4ELISA
Light Microscope (DMi1)Lucia391240General Cell Culture
Lipopolysaccaride (LPS)Invivogentlrl-eblpsmoDC Cell Culture
LIVE/DEAD Fixable Near-IR Dead Cell Stain KitThermo FischerL34975Flow Cytometry
MEM Non-Essential Amino Acids Solution (100x)Thermo Fischer11140050T Cell Culture
Penicillin-Streptomycin (100x)Thermo Fischer15140122General Cell Culture
Pipette ControllerVWR77575-370General Cell Culture
Rapamycin, 98+%Thermo FischerJ62473.MFmoDC Cell Culture
RPMI 1640 with GlutamaxThermo Fischer61870-036General Cell Culture
Separation BufferStem Cell Technologies20144Cell Separation
T Cell Stimulation Cocktail (500x)Thermo Fischer00-4970-93T Cell Culture
UltraComp eBead Plus Compensation BeadsThermo Fischer01-3333-41Flow Cytometry
Variable Pipette SetFischer Scientific05-403-152General Cell Culture

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