An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Summary

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Abstract

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not naïve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

Introduction

The blood-brain barrier separates the systemic circulation from the central nervous system (CNS)^1-3. It provides a physical barrier that inhibits the free movement of cells and the diffusion of water-soluble molecules and protects the brain from pathogens and potentially harmful substances. In addition to its barrier function, the BBB enables the delivery of oxygen and nutrients to the brain parenchyma, which ensures proper functioning of the neuronal tissue. Functional properties of the BBB are highly regulated by its cellular and acellular components, with highly specialized ECs being its main structural element. ECs of the BBB are characterized by the presence of tight junction (TJ) complexes, the lack of fenestrations, extremely low pinocytic activity, and permanently active transport mechanisms. Other components of the BBB the EC basement membrane, pericytes embedding the endothelium, astrocytic end feet and = associated parenchymal basement membrane also contribute to the development, maintenance and function of the BBB^2,4-6 and, together with neurons and microglia, form the neurovascular unit (NVU), which enables proper functioning of the CNS^7-9.

In a variety of neurological diseases, such as neurodegenerative, inflammatory or infectious diseases, the function of the BBB is compromised^2,5,10. Dysregulation of TJ complexes and molecular transport mechanisms leads to increased BBB permeability, leukocyte extravasation, inflammation and neuronal damage. In order to study BBB properties under such pathophysiological conditions, various in vitro BBB models have been established^9,11,12. Together they have provided valuable insights into the changes of barrier integrity, permeability as well as transport mechanisms. These models employ endothelial cells of human, mouse, rat, porcine or bovine origin^13-18; primary endothelial cells or cell lines are cultured either as a monoculture or together with pericytes and/or astrocytes in order to mimic more closely the BBB in vivo^19-25. In recent years, measurement of transendothelial electrical resistance (TEER) has become a widely accepted tool to assess endothelial barrier properties^26,27.

TEER reflects the impedance to the ion flux across the cell monolayer and its decrease provides a sensitive measure of compromised endothelial barrier integrity and hence increased permeability. Various TEER measurement systems have been developed, including Epithelial Voltohmmeter (EVOM), Electric Cell-substrate Impedance Sensing (ECIS), and real-time cell analysis^15,28-30. TEER reflects the resistance to the ion flux between adjacent ECs (paracellular route) and is directly proportional to the barrier integrity. In impedance spectroscopy^27,31, complex total impedance (Z) is measured, which provides additional information about the barrier integrity by measuring Ccl. Ccl relates to the capacitive current through the cell membrane (transcellular route): the cell layer acts like a capacitor in the equivalent electric circuit, separating the charges on both sides of the membrane and is inversely proportional to the barrier integrity. When grown on permeable inserts, ECs adhere, proliferate and spread over the microporous membrane. This resists the background capacitive current of the insert (which itself acts like a capacitor) and leads to a decrease in the capacitance until it reaches its minimal level. This is followed by the establishment of TJ complexes that seal off the space between adjacent ECs. This restricts the ion flux through the paracellular route, and TEER increases until it reaches its plateau. Under inflammatory conditions, however, the endothelial barrier is compromised: TEER decreases as TJ complexes get disrupted and Ccl increases as the capacitive component of the insert rises again.

Our TEER measurement uses the automated cell monitoring³² system: it follows the principle of impedance spectroscopy and extends its previous applications. Here, we describe an in vitro BBB model that enables the study of the barrier properties, including the interaction of brain endothelium with immune cells; in particular activated T cells. Such pathophysiological conditions are observed in autoimmune diseases of the CNS, such as multiple sclerosis and its animal model experimental autoimmune encephalomyelitis^33-37. Here, a crucial step is the transmigration of encephalitogenic, myelin-specific T cells across the BBB. This is followed by their reactivation in the perivascular space and entry into the brain parenchyma, where they recruit other immune cells and mediate inflammation and subsequent demyelination^1,35,38. However, molecular mechanisms of the interaction between such T cells and endothelial cells, the main constituents of the BBB, are not well understood. Our protocol aims to fill this gap and give new insights into the consequences on endothelial cells (i.e., barrier integrity and permeability) upon their direct contact and complex interplay with activated T cells.

The protocol described here makes use of primary mouse brain microvascular endothelial cells, grown as a monolayer on permeable inserts with microporous membranes. Endothelial cells are co-cultured with CD4⁺ T cells, which can be pre-activated either polyclonally or in an antigen-specific fashion. Co-culture of MBMECs with pre-activated, but not naïve T cells induces a decrease in TEER and an increase in Ccl, which provides a quantitative measure of the MBMEC dysfunction and barrier disruption. The technique is non-invasive: it uses built-in instead of chopstick electrodes, which prevent major disturbance of the EC monolayer; it can be used to monitor barrier function without the use of cell markers. It makes continuous measurements in an automated fashion and enables an independent assessment of the two barrier parameters (TEER and Ccl) simultaneously over time. The method is also sensitive enough to distinguish between different levels of T cell activation and effects of such T cells on ECs.

It can be used in a wide range of functional assays: different cytokines and/or chemokines implicated in inflammatory processes can be added to the co-culture of MBMECs and T cells; blocking antibodies against cell adhesion molecules on either the EC or T-cell side can be used; and inhibitors of T cell activation markers or of their cytolytic properties can be added during the T-cell priming or their co-culture with ECs. The assay is also useful for T-cell transmigration assays, as it can serve as a quality control of the MBMEC monolayer integrity prior to the addition of T cells. All this makes this method a versatile and reliable tool to study the BBB in vitro, with a focus on the effect of activated T cells on EC monolayer integrity. This is of particular importance for understanding the mechanisms of the BBB disruption in the pathogenesis of autoimmune diseases, such as MS and its animal model EAE, where self-reactive, encephalitogenic T cells cross the BBB and cause inflammation and neuronal damage.

Protocol

For all experiments, mice were bred and maintained under specific pathogen-free conditions in the central animal facility at the University of Münster, according to German guidelines for animal care. All experiments were performed according to the guidelines of the animal experimental ethics committee and approved by the local authorities of North Rhine-Westphalia, Germany (LANUV, AZ 84-02.05.20.12.217).

1. MBMEC Isolation and Culture

NOTE: Isolate MBMECs as previously described in detail¹⁴ with the following modifications:

1. Sacrifice 20 adult C57BL/6 mice (6-8 weeks old) by CO₂ inhalation and confirm their death by ascertaining cardiac and respiratory arrest.
2. Rinse the mouse with 70% ethanol and decapitate it using scissors; remove the scalp using forceps and scissors. Make incisions on the left and right side of the skull, starting from the foramen magnum. Lift the skull from its caudal side and take out the brain with forceps.
3. Under a laminar flow hood, use sterile forceps to place the brain in a Petri dish and remove the brain stem, cerebellum, and thalamus, keeping only the cortex.
   NOTE: Use of a microscope is not necessary for any of these steps.
4. Place the cortex onto a piece of sterile Western blotting paper and roll it gently with forceps until meninges are no longer visible.
5. After mechanical and enzymatic digestion (following the protocol¹⁴), collect endothelial cells from the density gradient by using a long, sterile needle and a 5 ml plunger. Endothelial cells appear as a murky layer above the red ring with erythrocytes. Transfer 20-25 ml of this layer into a new 50 ml centrifugation tube; top up the tube with DMEM.
6. After two rounds of washing¹⁴, resuspend the cells in 6 ml of MBMEC medium containing Puromycin (4 µg/ml) and seed them onto six coated wells of a 24-well plate; leave them in a tissue culture incubator at 37 °C and 5% CO₂ (referred to as 'incubator' from here on) for three days.
   NOTE: For details of MBMEC coating solution, see Materials Table.
7. Change the medium by adding 1 ml to each well of fresh MBMEC medium without Puromycin; put the plate back at 37 °C and 5% CO₂ for two more days.

2. Harvesting MBMECs

1. On day five after MBMEC isolation, pre-coat permeable inserts with MBMEC coating solution: add 80 µl of solution per insert and leave them at 37 °C and 5% CO₂ for 3 hr.
2. Carefully pipette out the coating solution and let the inserts air-dry for 30 min. Add 2 ml of room temperature (RT) PBS to each well of the plate, using MBMECs to wash the medium; repeat this step. Add 0.05% Trypsin-EDTA (300 µl per well) and leave the plate in the incubator for 5-10 min.
3. Add 2 ml of MBMEC medium to each well to stop trypsinization. Transfer the collected cells to a 15 ml centrifugation tube and centrifuge them at 700 x g for 8 min at 4 °C. Discard the supernatant and resuspend the cells in 1 ml of MBMEC medium without Puromycin.
4. Count the cells; mix 10 µl of cell suspension with 90 µl of 0.04% Trypan Blue (10x dilution of cells). Pipette 10 µl of this mix between the glass cover and the cell counting chamber (hemocytometer). Count Trypan Blue-free cells in all four quadrants of the chamber (N) and determine the average number of counted cells (N') as follows: N' = N/4.
5. Calculate the cell concentration (in 10⁶ cells/ml) using the following formula: C = N' x 10⁴ x 10, where 10⁴ is given by the dimensions of the hemocytometer and 10 is the dilution factor from step 2.4.
6. Seed 2 x 10⁴ cells in a volume of 260 µl per insert. Add 810 µl of MBMEC medium to the lower compartment of each well. Place the plate with inserts in the incubator until ready to proceed with section 4.
   NOTE: The volumes for the upper and lower compartment are insert-specific and do not work for all 24-well formats.

3. CD4⁺ T Cell Isolation and Stimulation

1. CD4⁺ T Cell Isolation
   1. Sacrifice one adult mouse (6-8 weeks old) by CO₂ inhalation and confirm its death by ascertaining cardiac and respiratory arrest.
   2. Place the mouse on its back on a clean dissection board and rinse it with 70% ethanol; use sterile scissors and forceps to open the peritoneum and remove the spleen and lymph nodes (inguinal, axillary, brachial and cervical); finally, transfer the tissue into a 15 ml centrifugation tube containing 5 ml of PBS on ice.
   3. Under the laminar flow hood, transfer the tissue by decanting PBS with the tissue into a 50 ml centrifugation tube with a 70 µm cell strainer on top; homogenize the tissue by pressing it with a 1 ml plunger through the strainer.
   4. Add 30 ml of FACS buffer and centrifuge it at 500 x g for 5 min at 4 °C. Discard supernatant and resuspend the cells in 10 ml of FACS buffer. Filter the suspension through a 40 µm cell strainer, add another 20 ml of FACS buffer to the tube.
   5. Centrifuge it at 500 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 500 µl of FACS buffer.
   6. Add 20 µl of mouse CD4 magnetic microbeads, mix well and incubate for 15 min at 4 °C. Add 25 ml of FACS buffer and centrifuge it at 500 x g for 5 min at 4 °C.
   7. In the meantime, place an LS separation column into the magnet and rinse it with 3 ml of FACS buffer. Discard the supernatant and resuspend the cells in 3 ml of FACS buffer.
   8. Add cells to the column. Wash the column with 3 ml of FACS buffer three times. Remove the column from the magnet and place it onto a new 15 ml centrifugation tube. Add 5 ml of FACS buffer, flush out the labelled cells with the column plunger and centrifuge it at 500 x g for 5 min 4 °C.
   9. Discard the supernatant and resuspend the cells in 3 ml of T cell medium. Count the cells as described in 2.4 and seed 1 x 10⁵ cells in 100 µl of medium per well.
2. CD4⁺ T Cell Stimulation
   1. Polyclonal CD4⁺ T cell stimulation
      1. Pre-coat a round-bottom 96-well plate with purified anti-mouse CD3 antibody (clone 145-2C11) in PBS, at a desired final concentration. For example, to pre-coat the full plate with α-CD3 at 1 µg/ ml, mix 10 µl of the antibody (stock concentration = 0.5 mg/ml) with 5 ml of PBS, vortex and add 50 µl of the mix to each well with a multichannel pipette.
      2. Leave the plate in the incubator for 3 hr. After isolating the T cells, wash the pre-coated plate twice with PBS. Add purified anti-CD28 antibody (clone 37.51) to isolated T cells at a desired final concentration (e.g., at 1 µg/ml); mix well. Seed the T cells and leave them in the incubator for two to three days.
   2. Antigen-specific CD4⁺ T cell stimulation with dendritic cells (DCs)
      NOTE: If DCs are used as antigen-presenting cells (APCs), follow the Protocol for T cell isolation, with these exceptions:
      1. Before homogenizing the spleen, inject it with 1 ml of Collagenase type IA in PBS at 0.5 mg/ml and transfer it to a 15 ml centrifugation tube.
      2. Incubate in the water bath at 37 °C for 15 min. After washing with PBS, resuspend the pellet in FACS buffer and add 20 µl of mouse CD11c magnetic microbeads, instead of CD4 microbeads.
      3. Use an MS separation column and the appropriate volumes: rinse the column with 1 ml of FACS buffer; resuspend cells in 1 ml of FACS buffer and wash the column with 1 ml of FACS buffer three times.
      4. Add antigen of choice to DCs (e.g., myelin oligodendrocyte glycoprotein (MOG) a.a. 35-55 at 20 µg/ml), mix well and seed 5 x 10⁴ cells in 100 µl of T cell culture medium per 96-round-bottom-well.
      5. Add 1 x 10⁵ T cells in 100 µl of T cell culture medium per 96-round-bottom-well at the end, as described in the Protocol for T cell isolation.
   3. Antigen-specific CD4⁺ T cell stimulation with B cells
      NOTE: If B cells are used as APCs, follow the Protocol for T cell isolation, with these exceptions:
      1. Use spleens from transgenic mice whose B cells are antigen-specific (e.g., IgH^MOG (Th) mice, whose B cells specifically recognize MOG_35-55).
      2. Use antigen-specific T cells (e.g., from TCR^MOG (2D2) mice). Add 20 µl of mouse CD19 magnetic microbeads instead of CD4 microbeads.
      3. Add the antigen of choice to the B cells (e.g., MOG_35-55 at 20 µg/ml), mix well and seed 5 x 10⁴ cells in 100 µl of B cell culture medium per 96-round-bottom-well.
      4. Add 1 x 10⁵ T cells in 100 µl of T cell culture medium per 96-round-bottom-well, as described in the Protocol for T cell isolation.

4. Setting Up and Performing TEER Measurement

1. Place the 24-well module of the TEER instrument under the laminar flow hood. Remove the lids and place inserts with MBMECs in the instrument using forceps.
2. Pipette 810 µl of fresh medium to the lower compartment of the module wells: add it carefully between the insert and the wall of the module well.
3. Close the lids and place the instrument in the incubator. Connect the instrument to its computer; turn on the instrument controller and open the software.
4. Select 'new measurement' in the pop-up window. Check 'show TEER' and 'show Ccl' boxes; then press 'start'. After completing the first measurement, select 'check all wells' in the 'results' tab to see all TEER and Ccl values.
5. Save the file: File>Save as>'the name of your file'.
   NOTE: As TEER and Ccl are continuously measured in an automated fashion, monitor their values over a period of three to five days. Changing the medium is not necessary, unless cell viability is suboptimal, as visualized by a lack of increase in TEER.
6. Choose the time point to co-culture MBMECs with T cells when Ccl is stable and lower than 1 µF/cm² and TEER has reached its maximum level.
7. Carefully inspect absolute TEER and Ccl values and exclude the wells in which MBMECs have not developed confluent enough monolayers by unchecking such wells.
8. Group the rest of the wells: right-click on a well and select 'add well to new average well'. Name it in the pop-up window; do the same for all individual wells to be grouped.
9. Check all average wells to confirm that all of them have the same initial conditions before the co-culture. If some have significantly different absolute TEER values or TEER slopes, or the standard errors are too large, redo the grouping.
   NOTE: The optimal grouping of wells provides minimal variation in TEER values, both within and between experimental groups.
10. In the 'experiment' tab, press 'pause', disconnect the instrument and take it out of the incubator. Remove the lids under the laminar flow hood.
11. Prepare pre-activated and/or naïve T cells, with or without specific cytokines, antibodies or other substances, as desired: For example: mix purified NA/LE rat anti-mouse IFN-γ antibody (clone XGM1.2) with prepared T cells, at 20 µg/ml per well of the TEER instrument.
    NOTE: If substances such as granzyme B inhibitor are used, they are added to T cell culture at the beginning of T cell stimulation: e.g., Granzyme B Inhibitor II (Calbiochem) is mixed with isolated T cells at a final concentration of 10 µM in DMSO. See Materials and Equipment for more details.
12. Remove some of the medium from the insert (the upper well compartment): e.g., carefully pipette out 150 µl (removing all the medium should be avoided as it could disturb the MBMEC monolayer).
13. Add T cells to MBMECs by carefully pipetting 150 µl of medium containing 2 x 10⁵ cells/insert.
    NOTE: When harvesting pre-activated T cells, count only blasting, Trypan Blue-free cells.
14. Close the lids and place the instrument back to the incubator. Reconnect the instrument and press 'resume measurement'. After 24 hr, press 'stop' in the 'experiment' tab and save the file (File>Save).

5. Data Export and Statistical Analysis

1. Export the results by choosing File>Export.
2. In the "settings" tab of the pop-up window, select ".dot" in the "decimal separator" option and "tabulator" in the "field delimiter" option.
3. In the "export results" tab, name the file, check all the wells to be exported, and check the "TEER" and "Ccl" boxes in the "choose data" option.
4. Press "export data," open the exported file, and copy the data to a spreadsheet.
5. Normalize the exported data (sorted by each replicate, with TEER and Ccl values given for each run (measurement)): Set TEER and Ccl values of the last time point before the co-culture to 100% and change accordingly the values for all other runs, relative to the '100%' run.
6. Copy normalized data to a software of choice to generate a graph, using individual wells and displaying the standard error of the mean for each treatment group.
7. Perform Two-way ANOVA statistical test with a Bonferroni correction for multiple comparisons, using the statistical software of choice.

Results

Figure 1 provides a general overview of the in vitro BBB model used to study the interaction between T cells and endothelial cells. The experiment consists of three major steps. The first step is the isolation of primary MBMECs from brain cortices, and their culture for five days. When they reach confluence in the cell culture plate, MBMECs are trypsinized and reseeded onto permeable inserts, which are then placed in the TEER instrument. The TEER and Ccl of MBMEC...

Discussion

Several steps of the described protocol are essential for a successful experiment. During the initial MBMEC isolation and culture, it is crucial that work is performed under sterile conditions as much as possible, to prevent the contamination of the cell culture with fungal spores or bacteria. In order to obtain a pure culture of ECs, it is recommended to use a medium containing Puromycin for the first three days, which enables survival of ECs, but not other cells types (especially pericytes)^41,42. Another cri...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
cellZscope	nanoAnalytics GmbH	www.nanoanalytics.com	including: 24-well Cell Module, Controller, PC with cellZscope software v2.2.2
Ultracentrifuge	Thermo Scientific	www.thermoscientific.com	SORVALL RC 6+; rotor F21S-8x50y; for MBMEC isolation
flow cytometer	Beckman Coulter	www.beckmancoulter.com	for analysis of T cell transmigration
FlowJo7.6.5 software	Tree Star	www.flowjo.com	for analysis of T cell transmigration
Oak Ridge centrifuge tubes, PC	Thermo Fisher Scientific	3118-0050	50 ml; for MBMEC isolation
Transwell membrane inserts - pore size 0.4 µm	Corning	3470	for TEER measurement as the main readout
Transwell membrane inserts - pore size 3 µm	Corning	3472	for TEER measurement as the quality control prior to T-cell transmigration assay
24-well cell culture plate	Greiner	650 180	flat-bottom; for MBMEC culture
96-well cell culture plate	Costar	3526	round-bottom; for immune cell culture
QuadroMACS Separator	Miltenyi Biotec	130-090-976	for T cell and B cell isolation; supports MACS LS columns
OctoMACS Separator	Miltenyi Biotec	130-042-109	for dendritic cell isolation; supports MACS MS columns
Neubauer counting chamber	Marienfeld	MF-0640010	for cell counting
Cell strainer, 70 µm	Corning	352350	for immune cell isolation
Cell strainer, 40 µm	Corning	352340	for immune cell isolation
MACS MultiStand	Miltenyi Biotec	130-042-303	for immune cell isolation
MACS LS separation columns	Miltenyi Biotec	130-042-401	for T cell and B cell isolation
MACS MS separation columns	Miltenyi Biotec	130-042-201	for dendritic cell isolation
Mouse CD4 MicroBeads	Miltenyi Biotec	130-049-201	for CD4+ T cell isolation
Mouse CD19 MicroBeads	Miltenyi Biotec	130-052-201	for B cell isolation
Mouse CD11c MicroBeads	Miltenyi Biotec	130-052-001	for dendritic cell isolation
Collagen type IV from human placenta	Sigma	C5533	for MBMEC coating solution
Fibronectin from bovine plasma	Sigma	F1141-5MG	for MBMEC coating solution
Collagenase 2 (CSL2)	Worthington	LS004176	for MBMEC isolation
Collagenase/Dispase (C/D)	Roche	11097113001	for MBMEC isolation
DNase I	Sigma	DN25	for MBMEC isolation
Fetal Bovine Serum (FBS)	Sigma	F7524	for MBMEC isolation
Bovine Serum Albumin (BSA)	Amresco	0332-100G	for MBMEC isolation
Percoll	Sigma	P1644-1L	for MBMEC isolation
DMEM (+ GlutaMAX)	Gibco	31966-021	for MBMEC isolation and MBMEC culture medium
Penicillin/Streptomycin	Sigma	P4333	for MBMEC isolation and MBMEC culture medium
Phosphate-Buffered Saline (PBS)	Sigma	D8537	for MBMEC and immune cell isolation
Heparin	Sigma	H3393	for MBMEC culture medium
Human Basic Fibroblast Growth Factor (bFGF)	PeproTech	100-18B	for MBMEC culture medium
Puromycin	Sigma	P8833	for MBMEC culture medium; only for the first three days
0.05% Trypsin-EDTA	Gibco	25300-054	for harvesting MBMECs
Collagenase Type IA	Sigma	C9891	for dendritic cell isolation
Trypan Blue solution, 0.4%	Thermo Fisher Scientific	15250061	for cell counting
EDTA	Sigma	E5134	for immune cell isolation
IMDM + 1% L-Glutamin	Gibco	21980-032	for T cell culture medium
X-VIVO 15	Lonza	BE04-418Q	protect from light; for B cell culture medium
β-mercaptoethanol	Gibco	31350-010	for B cell culture medium
L-Glutamine (100x Glutamax)	Gibco	35050-061	for B cell culture medium
mouse MOG35—55 peptide	Biotrend	BP0328	for antigen-specific T cell activation
purified anti-mouse CD3 Ab	BioLegend	100302	clone 145-2C11; for polyclonal T cell activation
purified NA/LE anti-mouse CD28 Ab	BD Pharmingen	553294	clone 37.51; for polyclonal T cell activation
Recombinant Murine IFN-γ	PeproTech	315-05	for T-cell transmigration assays
Recombinant Murine TNF-α	PeproTech	315-01A	for T-cell transmigration assays
NA/LE purified anti-mouse IFN-γ antibody	BD Biosciences	554408	clone XMG1.2; recommended final concentration: 20 µg/ml
Granzyme B Inhibitor II	Calbiochem	368055	recommended final concentration: 10 µM
PE anti-mouse CD4 antibody	Biolegend	116005	clone RM4-4; for analysis of T cell transmigration