Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.
Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/β-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.
The neurovascular unit (NVU) in the central nerve system (CNS) consists of the highly specialized microvascular endothelial cells (ECs), pericytes embedded in the endothelial basement membrane as well as the parenchymal basement membrane and astrocyte end-feet1. Within the NVU, brain microvascular endothelial cells (BMECs) are the key components that form the blood-brain barrier (BBB). BMECs form complex and continuous tight junctions and have extremely low pinocytotic activity compared to microvascular ECs in peripheral organs, which allow the BBB to inhibit the free paracellular diffusion of water-soluble molecules into the CNS. The expression of specific influx transporters and efflux pumps by BMECs ensures the uptake and export of nutrients and harmful molecules, respectively, from the CNS2. In addition, the BBB strictly controls immune cell entry into the CNS by expressing low levels of endothelial adhesion molecules crucial for immune cell trafficking into the CNS3. Under physiological conditions, the expression levels of adhesion molecules on the surface of BMECs, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are low, but these levels increase in some neurological disorders2. Morphological and functional breakdown of the BBB is reported in many neurological diseases, such as stroke4, multiple sclerosis (MS)5, and several neurodegenerative diseases6,7,8. Detailed investigation of the cellular and molecular characteristics of BMECs under both physiological and pathological conditions is an approach to identifying novel therapeutic strategies that target the BBB.
Until recently, primary or immortalized human and rodent BMECs were used to study the BBB. However, whether conclusions based on animal models of the BBB are readily applicable to the human BBB is unclear, since the expression of several important molecules, including adhesion molecules and solute carrier proteins, differs between humans and rodents9,10. Although human BMEC lines like hCMEC/D3 express appropriate levels of adhesion molecules11, these immortalized BMECs generally do not have complex tight junctions and robust barrier properties12. Primary human BMECs are useful to study barrier functions13, but they are not readily available to all researchers. Further, primary BMECs from patients can be difficult to obtain since they must be collected through a brain biopsy or surgery that is only performed under specific clinical conditions.
Recent advances in stem cell technology have allowed the differentiation of various human cell types, arising from stem cell sources like human induced pluripotent stem cells (hiPSCs). The hiPSC-derived models allow us to establish pathophysiological models using patient-derived samples. Several hiPSC-derived cell types can be combined to establish autologous co-cultures or organoids that better mimic physiological conditions. Several widely-used protocols14,15,16,17,18,19 may be used to differentiate hiPSC-derived BMEC-like cells that have robust diffusion barrier properties with the expression of BBB-specific transporters and efflux pumps, and are useful to study the paracellular diffusion of small molecules, molecular transport mechanisms, and drug delivery to the brain20,21. However, previous studies have shown that widely used hiPSC-derived BMEC-like cells lack the expression of key endothelial adhesion molecules, including VCAM-1, selectins, and ICAM-2, which are responsible for mediating interactions between immune cells and the BBB22. Furthermore, previous hiPSC-derived BMECs have been reported to display mixed endothelial and epithelial characteristics at the transcriptional level23. Therefore, we developed the extended endothelial cell culture method (EECM), a novel protocol that allows the differentiation of hiPSCs into BMEC-like cells that resemble primary human BMECs with respect to morphology, barrier characteristics, and endothelial adhesion molecule expression. This protocol describes the detailed methodological procedures to differentiate hiPSCs to BMEC-like cells displaying a mature immune phenotype.
Figure 1: Overview of the protocol. The manuscript presents a step-by-step protocol for differentiating hiPSCs into EECM-BMEC-like cells. The right schemes depict the cell populations at each step. Please click here to view a larger version of this figure.
The hiPSC line, HPS1006, was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan.
1. Induction of hiPSC differentiation into endothelial progenitor cells (EPCs)
2. Extended endothelial cell culture method (EECM) to differentiate brain microvascular endothelial cell-like cells (BMEC-like cells) and smooth muscle-like cells (SMLCs)
3. Validation of EECM-BMEC-like cells and SMLCs
Permeability assay
The permeability of sodium fluorescein was calculated by measuring the fluorescence intensity of the medium collected from the lower chamber at 15, 30, 45, and 60 min. A total of 150 µL of medium is sampled at each time point and the missing volume of 150 µL is replaced with hECSR medium. the fluorescence intensity is read using a fluorescent plate reader (485 nm excitation/530 nm emission) and the correct signals, clearance volumes, and permeabilities are calculated using a previously described formula18 (Table 2). It is recommended to confirm whether the fluorescence intensity of sodium fluorescein increases over time. Multiple filters-at least triplicates-should be used for one assay to ensure reproducibility. For healthy control-derived EECM-BMEC-like cells, the sodium fluorescein (376 Da) permeability should be below 0.3 x 10-3 cm/min. To confirm the formation of a confluent EECM-BMEC-like cell monolayer, immunofluorescence staining for junctional proteins of the EECM-BMEC-like cells of each filter used in the permeability assays should be performed following this assay.
Immunofluorescence staining
Immunofluorescence staining of EECM-BMEC-like cell junctional molecules, including claudin-5, occludin, and VE-cadherin1, was used to assess cell morphology and the presence of continuous and mature junctions (Figure 4). The monolayers of EECM-BMEC-like cells on the membranes of the filter inserts were fixed with cold methanol (-20 °C) for 20 s, blocked with blocking buffer (Table 1), and then incubated with primary and secondary antibodies. The EECM-BMEC-like cells exhibited spindle like shapes and zigzag shaped junctions, both of which are characteristic morphological features of BMECs27. Stimulation of the EECM-BMEC-like cells seeded on chamber slides with pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ) (0.1 ng/mL TNF-α + 2 IU/mL IFN- γ) diluted in SMLC-derived conditioned medium, upregulated the expression of adhesion molecules, such as ICAM-1 and VCAM-128 (Figure 5). Representative images of smooth muscle cell markers, including α-smooth muscle actin (SMA), calponin, and smooth muscle protein 22-Alpha (SM22a)29, are shown in Figure 6. SMLCs seeded on the chamber slide were fixed with 4% paraformaldehyde for 10 min, blocked with blocking buffer, and then incubated with primary and secondary antibodies.
Flow cytometry analysis of cell surface adhesion molecule expression by EECM-BMEC-like cells
Representative results for cell surface expression of endothelial adhesion molecules on EECM-BMEC like cells are displayed in Figure 7. Stimulation with pro-inflammatory cytokines, like TNF-α and INF-γ, upregulated the cell surface expression of several adhesion molecules, including ICAM-1, VCAM-1, and P-selectin. Cultivating EECM-BMEC-like cells with SMLC-conditioned medium enhanced endothelial VCAM-1 cell surface expression. The effect of the induction of VCAM-1 cell surface expression may vary between batches of SMLC-conditioned medium. It is recommended that several batches of conditioned medium harvested from SMLCs, derived from the same hiPSC source, be stored when differentiating SMLCs, in order to verify which batch induces the appropriate expression of VCAM-1.
Immune cell adhesion assay under static conditions
The number of attached immune cells correlated to the expression level of functional adhesion molecules on the surface of EECM-BMEC-like cells. Stimulation with inflammatory cytokines upregulated the expression of endothelial adhesion molecules and promoted the increased number of immune cells that adhered to EECM-BMEC-like cell monolayers (Figure 8). The current experiment demonstrated the functionality of adhesion molecules on EECM-BMEC-like cells, making this model suitable for studying immune cell-EC interactions.
Figure 2: Purification of CD31+ ECs. Dot plots of representative flow cytometry data from scatter gating of ECs and FITC-labeled CD31 staining of cell populations before (step 1.4.7) and after (step 1.4.14) MACS. MACS improves the purity of CD31+ EPCs in the population. Abbreviations: SSC = side scatter; FSC = forward scatter; FITC = fluorescein isothiocyanate; MACS = magnetic activated cell sorting. Please click here to view a larger version of this figure.
Figure 3: EECM-BMEC-monolayer permeability (10-3 cm/min) calculated from the raw fluorescence intensity of sodium fluorescein. The linear slope of clearance volume is calculated using linear regression for each filter (Figure 3A). The permeability of sodium fluorescein is calculated using two formulas (Figure 3B). (A) The linear slope of clearance volume versus time was calculated using linear regression for filter 1 (mc1) and the blank filter (mf). The mc1 and mf are coefficients of Xc1 and Xf, respectively. (B) Formula for calculating fluorescein permeability (Pe) using mc and mf (Formula 1). Pe units were converted using the surface area of a filter (Formula 2). Please click here to view a larger version of this figure.
Figure 4: EECM-BMEC-like cells display mature cellular junctions. Immunofluorescence staining for claudin-5, occludin, or VE-cadherin (red) in EECM-BMEC-like cells grown on membranes of insert filters. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (blue). Staining was performed on the exact same filter inserts used for the permeability assays. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 5: Expression of endothelial adhesion molecules by EECM-BMEC-like cells. Immunofluorescence staining was performed on EECM-BMEC-like cells grown on membranes of filter inserts in the presence of SMLC-derived CM. Immunostaining for ICAM-1 or VCAM-1 (red) is shown for non-stimulated and 1 ng/mL TNF-α + 20 IU/mL IFN- γ stimulated EECM-BMEC-like cells. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 6: Characterization of SMLCs. Immunocytochemistry of α-smooth muscle actin (SMA), calponin, or smooth muscle protein 22-Alpha (SM22a) (red) for SMLCs grown on chamber slides is shown. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 7: Endothelial cell surface expression of adhesion molecules on EECM-BMEC-like cells. Results of flow cytometry analysis of EC surface adhesion molecule expression on EECM-BMEC-like cells is shown. EECM-BMEC-like cells were cultured using SMLC-derived conditioned medium. Blue, red, and gray lines of the histogram overlays show the non-stimulated (NS) condition, 1 ng/mL TNF-α + 20 IU/mL IFN-γ-stimulated condition, and isotype control, respectively. The cell surface expression of endothelial adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), P-selectin, E-selectin, CD99, and platelet endothelial cell adhesion molecule-1 (PECAM-1) were assessed. Please click here to view a larger version of this figure.
Figure 8: Adhesion of immune cells on EECM-BMEC-like cells. (A) Images of fluorescently labeled adherent immune cells on non-stimulated (NS) and 0.1 ng/mL TNF-α + 2 IU/mL IFN-γ-stimulated (TNF-α + IFN-γ) EECM-BMEC-like cell monolayers. The images correspond to the centers of the wells. Scale bar = 50 µm. (B) The number of fluorescently labeled immune cells on monolayers of NS and TNF-α + IFN-γ-stimulated EECM-BMEC-like cells. Adherent immune cells/fields of view (FOVs) were automatically counted using FIJI software. Dots represent the number of attached T cells. Bars show the mean value, and error bars show the standard deviation (SD) of eight trials. Please click here to view a larger version of this figure.
Table 1: Details of specific reagents for the assays. The name and exact amount of ingredients for each specific reagent are described. Please click here to download this Table.
Table 2: Example of raw data of the fluorescence plate reader for Pe calculation. Numbers in boldface type are the raw fluorescence intensity of sodium fluorescein measured by a plate reader. In order to accurately analyze the data, it is necessary to remove the background signal from the raw values and account for any signal loss resulting from sampling the bottom chamber, and subsequently correct the signal. For example, after subtracting the background, the 15 min sample exhibits a signal of 100 relative fluorescence units (RFU), and the 30 min sample exhibits a signal of 150 RFU. The corrected signal at 30 min is (150 RFU + the missing values at 15 min [100 RFU x 150 µL/1,500 µL]), which is 150 RFU + 10 RFU = 160 RFU. The clearance volume = (1,500 x [SB,t])/(ST,60 min), where 1,500 is the volume of the bottom chamber (1,500 µL), SB,t is the corrected signal at time t, and ST,60 min is the signal of the top chamber at 60 min. Please click here to download this Table.
Critical points and troubleshooting
Before starting EPC differentiation, researchers should ensure that no spontaneous cell differentiation events have occurred in the hiPSC cultures. The absence of spontaneously differentiated cells and the use of pure hiPSC colonies is critical for obtaining reproducible results. The hiPSC seeding density on Day -3 is important for obtaining a high purity of CD31+ EPCs after MACS. The seeding density for each hiPSC line and each passage may require optimization. Depending on the hiPSC line and passage number, the seeding density can range between 75 x 103 to 400 x 103 hiPSCs per well of a 12-well plate (20-100 x 103/cm2). The minimum density checkpoint of hiPSCs is the cell density on Day 2. The hiPSCs should reach 100% confluency by Day 2 at the latest. If the hiPSCs are not confluent by Day 2, the purity of CD31+ EPCs after MACS will usually be quite low. In this case, the hiPSC seeding density can be increased. If large numbers of differentiating cells detach from the plate around Day 3 to Day 5, the initial hiPSC seeding density can be decreased. The 7-8 µM CHIR99021 in our experience is the optimal concentration for the hiPSCs lines used here, but the concentration may need to be optimized for other hiPSC lines that may respond differently to the inhibitor treatment. The purity of CD31+ EPCs should be confirmed before and after MACS. Before continuing MACS, the pre-sorted cell mixture should be >10% CD31+ cells. CD31+ cell percentages of <6% typically result in <80% EPCs after MACS. Optimization of the initial seeding density and/or the CHIR99021 concentration is needed in this situation.
For successful selective passaging and generation of pure EC monolayers, the post-MACS purity of CD31+ EPCs is critical. If the purity after MACS is <90%, one or two additional washes are recommended (steps 1.4.11-1.4.12). Ideally, the post-MACS purity should be >95%. The EPC seeding density on collagen-coated plates should be optimized according to the hiPSC line to achieve 100% confluency within 3-7 days. Waiting until the ECs are 100% confluent will usually lead to successful selective passage. However, even for 100% confluent ECs, some hiPSC lines' SMLCs also detach early. In this case, selective passaging at lower confluency (e.g., ≤80%) may be effective. If some SMLCs detach earlier than ECs, the ECs often cannot be rescued from the mixed EC-SMLC population. In this case, shortening the activation time for the dissociation reagent in passaging ECs and repetitive selective passaging may be helpful. The use of a commercial dissociation reagent rather than trypsin as a dissociation reagent is beneficial for selective passaging, as trypsin does not allow the separate detachment of ECs and SMLCs. Our permeability assays using small molecule tracers and testing of tight junction and adhesion molecule expression levels indicate that EPCs, EECM-BMEC-like cells, and SMLCs can be stored in liquid nitrogen for at least 2 years.
Significance and limitations of the method
The method differentiates CD31+ EPCs from hiPSCs through the use of chemical GSK-3 inhibitors to activate Wnt/β-catenin signaling. After positive selection of CD31+ EPCs by MACS, EPCs are cultured in a defined endothelial medium that promotes differentiation into mixed endothelial and SMLC populations. Selective passaging of these mixed populations with different adhesive properties allows the separation of ECs from SMLCs. After one or two passages, EECM-BMEC-like cells exhibit barrier properties and the expression of endothelial adhesion molecules that recapitulate those of primary human BMECs. Co-culture with SMLCs or their supernatants induces the cytokine-induced expression of VCAM-1.
In vivo, the BBB maintains CNS homeostasis by establishing low paracellular and transcellular permeability of molecules, through the transport of nutrients via specific transporters and the control of immune cell trafficking into the CNS. For studies of the BBB, a suitable model that displays the respective molecules and functions of interest is essential. Production of EECM-BMEC-like cells using defined reagents and samples from patients or healthy subjects provides a scalable human BBB model. The advantages of a model using EECM-BMEC-like cells over other BBB models are: 1) a morphology and endothelial transcriptome profile30 that resembles that of primary human BMECs; 2) the presence of mature tight junctions; 3) desirable barrier properties; and 4) the robust expression of endothelial adhesion molecules, including ICAM-1, ICAM-2, VCAM-1, E- and P-selectin, CD99, melanoma cell adhesion molecule (MCAM), and activated leukocyte cell adhesion molecule (ALCAM)22. Thus, this model is particularly useful for studying interactions between immune cells and BMECs. Although the permeability of small molecule tracers is higher for EECM-BMEC-like cells than that previously reported for iPSC-derived BMEC-like cells14,15, the barrier properties do compare quite well to those described for primary human BMECs. This similarity indicates that EECM-BMEC-like cells are likely to be a good in vitro model of the BBB. E-selectin expression on EECM-BMEC-like cells under physiological conditions must be taken into account when using this model to study non-inflamed BBBs that lack constitutive E-selectin expression in vivo31. In our previous study, we demonstrated that EECM-BMEC-like cells could phenocopy the BBB, as observed in the brains of MS patients with respect to disrupted tight junctions. This results in a higher permeability of small molecules and increased expression of functional adhesion molecule, mediating the increased adhesion and transmigration of immune cells across the BMEC-like cells32. Furthermore, we showed that the activation of Wnt/β-catenin signaling can ameliorate the disruption of tight junctions and increased VCAM-1 expression in MS-derived EECM-BMEC-like cells32. These results indicate that the model is indeed useful to study the role of the BBB in neuroimmunological diseases, such as MS.
Taken together, EECM-BMEC-like cells are a promising tool for in-depth understanding of pathophysiological mechanisms at the level of the BBB and as a tool to develop new therapeutic targets for BBB stabilization. In the future, the model can be applied to study BBB dysfunction in a wider spectrum of diseases and could open avenues for novel therapeutic approaches.
The authors have nothing to disclose.
HN was supported by the Uehara Memorial Foundation, an ECTRIMS Postdoctoral Research Exchange Fellowship, JSPS under the Joint Research Program implemented in association with SNSF (JRPs) Grant No. JPJSJRP20221507 and KAKENHI Grant No. 22K15711, JST FOREST Program (Grant Number JPMJFR2269, Japan), YOKOYAMA Foundation for Clinical Pharmacology Grant No. YRY-2217, the ICHIRO KANEHARA FOUNDATION, the Narishige Neuroscience Research Foundation, the NOVARTIS Foundation (Japan) for the Promotion of Science, and the YAMAGUCHI UNIVERSITY FUND. BE was supported by the Swiss MS Society and the Swiss National Science Foundation (grants 310030_189080 and ZLJZ3_214086) and Strategic Japanese-Swiss Science and Technology Programme (SJSSTP) grant IZLJZ3_214086.
0.22 mm Syringe filter | TPP | 99722 | |
15 mL Centrifuge tube | Falcon | 352196 | |
40 μm Falcon cell strainer | Falcon | 352340 | |
5 mL Round-bottom tube | SPL | 40005 | |
50 mL Centrifuge tube | Falcon | 352070 | |
96-Well plate, round bottom | SPL | 34096 | |
Accutase | Sigma-Aldrich | A6964-500ml | |
Acetic acid | Sigma-Aldrich | 695092 | |
Advanced DMEM/F12 | Life Technologies | 12634 | |
All-in-One Fluorescence Microscope | Keyence | BZ-X810 | |
B-27 Supplement (503), serum free | Thermo Fischer Scientific | 17504044 | |
Bovine serum albumin (BSA), 7.5% in dPBS | Sigma-Aldrich | A8412 | |
CellAdhere Dilution Buffer | STEMCELL Technologies | ST-07183 | |
CellTracker Green CMFDA Dye | Invitrogen | C7025 | |
Chamber slides | Thermo Fischer Scientific | 178599 | |
CHIR99021 | Selleck Chemicals | S1263 | |
Collagen IV from human placenta | Sigma-Aldrich | C5533 | |
Corning tissue culture plates (12-well) | Corning | 3512 | |
Corning tissue culture plates (6-well) | Corning | 3506 | |
Cryo tube innercap 2.0 mL | Watson | 1396-201S | |
Dimethylsulfoxide (DMSO), sterile | Sigma-Aldrich | D2650 | |
DMEM (13), [+] 4.5 g/L D-glucose, [-] L-glutamine, [-] pyruvate | Thermo Fischer Scientific | 31053-028 | |
Dulbecco’s (d) PBS (without calcium, magnesium) | Thermo Fisher | 14190250 | |
Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM-F12) | Thermo Fischer Scientific | 11320074 | |
EasySepFITC Positive Selection Kit II | STEMCELL Technologies | 18558 | |
EasySepMagnet | Stemcell Technologies | 18000 | |
Ethylenediaminetetraacetic Acid Solution0.02% in DPBS | Sigma | E8008-100ML | |
Fetal Bovine Serum, qualified | Thermo Fischer Scientific | 10270106 | |
Fibronectin from bovine plasma | Sigma-Aldrich | F1141 | |
Ficoll-Paque PLUS | Sigma-Aldrich | GE17144002 | |
FIJI software (Version 2.0.0) | Image J, USA | ||
FlowJo version10 | BD | ||
Fluorescein sodium salt | Sigma-Aldrich | F6377 | |
GlutaMAX Supplement | Thermo Fischer Scientific | 35050-061 | |
Glutaraldehyde solution | Sigma-Aldrich | G6257 | |
HCL | Sigma-Aldrich | H1758 | |
HEPES buffer solution | Thermo Fischer Scientific | 15630-056 | |
Human Endothelial Serum Free Medium (hESFM) | Thermo Fischer Scientific | 11111-044 | |
Human fibroblast growth factor 2 (FGF2) | Tocris | 233-FB-500 | |
iPS human induced pluripotent stem cells | Riken RBC | HPS1006 | |
Kanamycin Sulfate (100x) | Thermo Fischer Scientific | 15160-047 | |
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | Sigma-Aldrich | A8960-5G | |
L-Glutamine 200 mM (1003) | Thermo Fischer Scientific | 25030-024 | |
Matrigel, growth factor reduced | Corning | 354230 | |
MEM NEAA (1003) | Thermo Fischer Scientific | 11140-035 | |
Methanol | Sigma-Aldrich | 32213 | |
Mowiol | Sigma-Aldrich | 81381 | |
mTeSR Plus-cGMP | STEMCELL Technologies | ST-100-0276 | |
mTeSR1 complete kit (basal medium plus 53 supplement) | STEMCELL Technologies | 85850 | |
NaCl | Sigma-Aldrich | 71376 | |
Paraformaldehyde | Millipore | 104005 | |
Pen Strep | Thermo Fischer Scientific | 15140-122 | |
Recombinant Human IFN-gamma Protein | R&D Systems | 285-IF-100 | |
Recombinant Human IL-2 | BD Biosciences | 554603 | |
Recombinant Human TNF-alpha Protein | R&D Systems | 210-TA-020 | |
ROCK inhibitor Y-27632 | Tocris | 1254 | |
RPMI medium 1640 | Thermo Fischer Scientific | 21875-034 | |
Scalpel | FEATHER | 2975-11 | |
Skim milk | BD Biosciences | 232100 | |
Sodium azide (NaN3) | Sigma-Aldrich | 71290 | |
Sodium pyruvate | Thermo Fischer Scientific | 11360-039 | |
Transwells, PC Membrane, 0.4 mm, 12 mm, TC-Treated | Corning | 3401 | |
Tris base | Sigma-Aldrich | 93362 | |
Triton X-100 | Sigma-Aldrich | X100 | |
Vitronectin XF | STEMCELL Technologies | 078180 | |
Water, sterile, cell culture | Sigma-Aldrich | W3500 |