A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Summary

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an αNFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Introduction

Chronic lymphocytic leukemia (CLL) represents the most common leukemia in adults in western countries, exhibiting distinct accumulation of CD19, CD23, and CD5 expressing mature B cells¹. Most patients exhibit an indolent disease course, which does not necessitate specific treatment for many years. In contrast, some patients show rapid progression requiring immediate therapeutic interventions with immune-chemotherapy or other targeted therapies^2,3. Nuclear factor of activated T cells (NFAT) is a family of transcription factors controlling various developmental and activation processes in numerous cell types^4,5,6. We recently demonstrated overexpression and constitutional activation of NFAT2 in CLL cells from patients with indolent disease⁷. Here, it regulates an unresponsive state to B cell receptor stimulation called anergy⁷. To demonstrate that NFAT2 binds to the lymphocyte-specific protein tyrosine kinase (LCK) promoter and regulates LCK expression in human CLL cells, a specific chromatin immunoprecipitation assay (ChIP) was developed and employed.

ChIP is one of the several techniques to investigate the role of transcription factors in gene expression⁸. Gene expression is tightly orchestrated in a very complex manner by several regulators with transcription factors taking an irreplaceable part in this process^9,10,11,12. Transcription factors regulating the gene expression in a spatial and temporal context have been identified in numerous species (e.g., for development and differentiation)^{13,14,15,16,17,18}. Errors in the intricate control mechanisms involving transcription factors can lead to a variety of pathologic processes including cancer^19,20. Hence, identification of transcription factors and their respective targets might offer novel therapeutic avenues^21,22. To investigate this intriguing field several methods are available like ChIP, electrophoretic mobility shift assay (EMSA), various DNA pull-down assays and reporter-assays^{8,11,12,23,24}.

To demonstrate that a certain transcription factor interacts with specific regions of the genome in vivo, ChIP is an ideal technique²⁵. For this purpose, DNA and associated proteins in living cells are cross-linked using UV irradiation or formaldehyde (cross-linked ChIP, XChIP). This step is omitted to obtain better DNA and protein recovery in the so-called native ChIP (NChIP)²⁶. The DNA-protein complexes are subsequently sheared by sonication into fragments of approximately 200-500 base pairs (bp) and immunoprecipitated from the cell debris using a specific antibody against the transcription factor of interest. The associated DNA fragments are then purified and characterized by PCR, molecular cloning, and sequencing. Alternative techniques use microarrays (ChIP-on-Chip) or the next-generation sequencing (ChIP-Seq) to analyze the immunoprecipitated DNA.

ChIP was first introduced by Gilmour and Lis in 1984 when they used UV light to covalently cross-link DNA and bound proteins in living bacteria²⁷. Upon cell lysis and immunoprecipitation of bacterial RNA polymerase, specific probes of known genes were used to map the in vivo distribution and density of RNA polymerase. The method was subsequently used by the same investigators to analyze the distribution of eukaryotic RNA polymerase II on heat shock protein genes in Drosophila²⁸. The XChIP assay was further refined by Varshavsky and coworkers who first used formaldehyde cross-linking to study the association of histone H4 with heat shock protein genes^29,30. The NChIP approach, which carries the advantage of a better DNA and protein recovery due to naturally intact epitopes and, therefore, greater antibody specificity, was first described by Hebbes and colleagues in 1988³¹.

The advantage of ChIP in comparison to other techniques to analyze DNA-protein interactions is in fact, that the actual interaction of a transcription factor can be investigated in vivo and no probes or artificial conditions created by buffers or gels are employed^8,11,12. By combining ChIP with next-generation sequencing, multiple targets can be identified simultaneously.

Major limitations of this technique are its limited applicability to large-scale assays in intact organisms²⁵. The analysis of differential gene expression patterns can also be challenging using ChIP techniques if the respective proteins are expressed only at low levels or during narrow time windows. Another potentially limiting factor is the availability of an appropriate antibody suited for ChIP¹¹.

The ChIP protocol presented here can be employed for the in vivo identification of target genes of a transcription factor by quantitative real-time PCR (qRT-PCR). Specifically, the goal was to identify novel target genes of NFAT2 in CLL. ChIP was chosen because of its potential to directly demonstrate the binding of NFAT2 to the promoter regions of different target genes under natural conditions in human CLL patient cells.

Protocol

All experiments conducted with human material were approved by the Ethics Committee of the University of Tübingen and written informed consent was obtained from all patients who contributed samples to this study.

1. Isolation and Stimulation of Jurkat cells

NOTE: To optimize the protocol, use the Jurkat cell line which is known to express the high levels of NFAT2. All steps are performed under a laminar flow hood.

1. Prepare 50 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin by warming it to 37 °C in a water bath.
2. Culture Jurkat cells for 1-2 d in a cell culture flask at 37 °C and 5% CO₂ in 20 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin to a density of approximately 2 x 10⁶ cells/mL (cells are counted with Trypan blue staining and a Neubauer chamber under the microscope) and harvest them by centrifuging for 5 min at 200 x g.
3. Remove the supernatant with a pipette and resuspend the cells in 10 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin. Subsequently, count the cells with a conventional trypan blue staining and a Neubauer chamber under the microscope.
4. Centrifuge the cells from step 1.3 for 5 min at 200 x g and remove the supernatant by aspiration. Then, resuspend the cells at a density of 2 x 10⁷ cells/mL in RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin and transfer 1 mL in a new conventional 5 mL tube by pipetting.
5. Stimulate the cells by adding 32.4 nM phorbol 12-myristate 13-acetate (PMA) and 1 µM ionomycin. Then incubate the cells for 16 h at 37 °C and 5% CO₂ with the lid of the tube opened (to avoid contamination, a sealing film permeable to air can be used).

2. Isolation and Stimulation of Primary CLL Cells from Human Patients

NOTE: Patient samples were acquired and stimulated as previously described⁷. All steps are performed under a laminar flow hood.

1. Prepare the equipment necessary to draw blood from patients and to perform a density gradient separation: 21-G needles, tourniquet, NH₄-heparin-blood collection syringes (e.g., S-monovettes), 1x PBS, and density gradient medium (density 1.077 g/mL).
2. Upon venipuncture, draw the blood from CLL patients (ca. 40 mL blood per patient) into the syringes. Then transfer the blood in 50 mL conical tubes. Wash the syringe with 10 mL PBS and pool the blood-PBS-suspension in the 50 mL tubes (adjust the number of tubes to the volume of blood).
3. Prepare 15 mL of the density gradient medium in a fresh 50 mL conical tube. Subsequently, layer 35 mL of the blood-PBS-suspension carefully on the density gradient medium. For this purpose, hold the tube at a flat angle and slowly pipette the blood-PBS-suspension against the bottom wall of the 50 mL conical tube. As more of the blood-PBS-suspension is accumulating in the 50 mL conical tube, increase the angle of the tube up to a right angle. Repeat this step according to the volume of the blood-PBS-suspension.
4. Perform centrifugation at 560 x g for 20 min (without brake), then extract the mononuclear cell phase which contains the peripheral blood mononuclear cells (PBMCs). To do so, collect the white interphase of the density gradient separation with a 5 mL serological pipette and transfer it to a new 50 mL conical tube.
5. Fill the cell-suspension to 50 mL with PBS and centrifuge for 5 min at 360 x g. Remove the supernatant by aspiration. Repeat this step with centrifugation for 5 min at 275 x g. Then, pool the cells of one patient in 5-10 mL of PBS in a 50 mL conical tube.
6. Count the cells as described in 1.3.
   NOTE: It depends on the proportion of CLL cells in the isolated PBMCs, if the cells can be used directly for this procedure or a B cell isolation has to be conducted, e.g., via magnetic cell sorting (MACS). The B cell isolation is recommended but can be omitted if the proportion of CLL cells is above 95% of total lymphocytes (determined via flow cytometry). Blood of CLL patients is fixed and lysed according to the manufacturer's instructions. Then a staining with CD19-FITC and CD5-PE antibodies is performed according to the manufacturer's instructions and the cells are analyzed via flow cytometry. One exemplary patient is shown in Figure 1, in which the proportion of CLL cells is 89.03%. Thus, a B cell isolation has to be performed in this patient. The proportion of physiological B cells is negligible (3.72% in the example).
7. Wash the cells with 10 mL of pre-warmed (37 °C) RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 100 mM non-essential amino acids, 1 mM sodium pyruvate and 50 mM β-mercaptoethanol for 5 min at 200 x g and remove the supernatant by aspiration.
8. Adjust the cells to 2 x 10⁷ cells/mL in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 100 mM non-essential amino acids, 1 mM sodium pyruvate and 50 mM β-mercaptoethanol (37 °C) and fill 1 mL of the cell suspension into a 5 mL tube.
   NOTE: The ß-mercaptoethanol is employed to counteract oxidative stress.
9. Stimulate the cells by adding 32.4 nM PMA and 1 µM ionomycin. Then incubate the cells for 16 h at 37 °C and 5% CO₂ with the lid of the tube opened (to avoid contamination, a sealing film permeable to air can be used).
   NOTE: To reduce the level of stress the cells can be rested for 1 h at 37 °C and 5% CO₂ in the incubator prior to the 16 h of stimulation.

3. Fixation, Cell Lysis, and Chromatin Shearing

NOTE: Patient samples and Jurkat cells are fixed and lysed with a commercially available ChIP kit according to the manufacturer's instructions with modifications as described previously⁷. The fixation is performed under a laminar flow hood.

1. Prepare a 1.5 mL tube with 1 mL of 37% formaldehyde, a 1.5 mL tube with 1 mL of 1.25 M glycine, a 5 mL tube with 4 mL of 1x PBS and a box with ice for the fixation of the cells.
2. After stimulation of the cells for the respective incubation time in step 1.5 or 2.9, spin the 5 mL tubes at 200 x g for 5 min and resuspend the cells in 500 µL of PBS in the 5 mL tubes.
3. Add 340 µM formaldehyde (13.5 µL of 37% formaldehyde) to the cells. After brief mixing by carefully pipetting up and down incubate the suspension for 2.5 min (Jurkat cells) or 5 min (patient sample) at room temperature.
   NOTE: Prolonged fixation time is needed for primary cells to assure optimal shearing.
4. Add 125 mM glycine (57 µL of 1.25 M glycine) to stop the fixation and incubate for another 5 min. Put the cells on ice immediately thereafter and centrifuge at 500 x g for 5 min at 4 °C. Remove the supernatant by aspiration.
5. Wash the cells twice with 1 mL of ice-cold PBS at 200 x g for 5 min at 4 °C and remove the supernatant every time by aspiration.
   NOTE: After the fixation, the protocol can be paused. Fixed cells should be stored at -80 °C. To test, if the epitope of the antibody intended to use in the following protocol is not masked via fixation, additional samples can be used for analysis via SDS-PAGE gel electrophoresis and Western blot. These steps are performed with 100 µg of protein according to manufacturer's instructions. All αNFAT2 antibodies are used at a dilution of 1:1000, the GAPDH antibody at a dilution of 1:10000. An exemplary image of fixed samples from Jurkat cells with appropriate and inappropriate antibodies is shown in Figure 2.
6. Resuspend the cell pellet in 5 mL of commercially available lysis buffer 1 by pipetting up and down. Then, place the samples on the ice on a shaker and incubate them for 10 min while shaking.
   NOTE: Prior to lysis, the cells can be disintegrated for 10 s in liquid nitrogen. This is useful for Jurkat cells but should be avoided in primary patient samples. For the disintegration, place the 5 mL tubes for 5 s in 5-10 mL of liquid nitrogen in a specific container. Wear safety glasses and appropriate safety gloves.
7. Subsequently, centrifuge the tubes at 500 x g for 5 min at 4 °C and remove the supernatant carefully by pipetting.
8. Homogenize the cells in 5 mL of commercially available lysis buffer 2 by pipetting up and down and incubate for 10 min on ice while shaking. Then centrifuge the tubes at 500 x g for 5 min at 4 °C and remove the supernatant carefully by pipetting.
9. Resuspend the cell pellet in 500 µL (Jurkat cells) or 140 µL (patient samples) of shearing buffer 1 containing 1x protease inhibitor (5 µL or 1.4 µL, respectively) and incubate the mixture for 10 min on ice.
10. For chromatin shearing, transfer 140 µL of the cell-suspension from step 3.9 into sonicator tubes (avoid producing air bubbles and repeat this step if necessary due to sample volume). Place the tubes in the focused ultra-sonicator at a temperature of 7 °C and shear for 10 min (cell line) or 7.5 min (patient samples) with an average incident power of 9.375 W to obtain 200-500 bp DNA fragments.
11. Finally, centrifuge at 15700 x g for 10 min at 4 °C in the small centrifuge and collect the supernatant in a new 1.5 mL tube.
12. To test for the appropriate fragment sizes, analyze 20 µL of the sheared chromatin via gel-electrophoresis in 1.5% TBE agarose gel. An exemplary image of sheared chromatin from Jurkat cells of good and bad quality is shown in Figure 3. At this point, the protocol can be potentially paused. Sheared chromatin should be stored at -80 °C.

4. Chromatin Immunoprecipitation

NOTE: The chromatin immunoprecipitation was performed with a commercially available ChIP kit according to the manufacturer's instructions with modifications⁷.

1. Calculate the number of immunoprecipitations (70 µL (Jurkat cells) or 15 µL (patient samples) of sheared chromatin plus one antibody) and prepare 20 µL of protein A coated beads per precipitation in a 1.5 mL tube. Wash the beads in 40 µL of 1x ChIP buffer 1 per precipitation by pipetting up and down, let the beads rest in a magnetic rack for 1 min and remove the supernatant by pipetting. Perform this step four times in total. Eventually, resuspend the beads in the original volume with 1x ChIP buffer 1.
2. For the immunoprecipitation itself, use 10 µg of the αNFAT2 antibody (7A6) to capture NFAT2 with its bound target sequences. Use 2.5 µg of a rabbit IgG antibody (DA1E) as an IgG-control. Prepare the reactions in fresh 1.5 mL tubes as indicated in Table 1.
   NOTE: It is important to use a monoclonal antibody with high affinity whose epitope is not masked during fixation for this protocol. Polyclonal antibodies introduce an additional level of variation making it significantly more difficult to appropriately interpret the received results.
3. Incubate the mixture from step 4.2 overnight at 4 °C on a rotating wheel at 6 rpm.
4. On the next day spin the tubes for 5 s at 7,000 x g in the small centrifuge, incubate them in a magnetic rack for 1 min and remove the supernatant by aspiration. Wash the beads once with each of the wash buffers 1, 2, 3 and 4 consecutively.
5. To do so, resuspend the beads in 350 µL of wash buffer and incubate them for 5 min at 4 °C on a rotating wheel at 6 rpm.
6. Thereafter, spin the tubes for 5 s at 7000 x g in the small centrifuge. Then, place them for 1 min in a magnetic rack, remove the supernatant and add the next wash buffer.
7. After the last washing step, remove the supernatant by pipetting, take up the beads in 100 µL of elution buffer 1 and incubate them for 30 min on a rotating wheel at 6 rpm.
8. Spin the tubes shortly (5 s at 7000 x g in the small centrifuge) and place them in a magnetic rack for 1 min.
9. Then transfer the supernatant by pipetting to a new 1.5 mL tube and add 4 µL of elution buffer 2.
10. To create the input control, mix 1 µL of the sheared chromatin with 99 µL of elution buffer 1 and 4 µL of elution buffer 2 (in this setup, there are three tubes per patient: one input control, one IgG-control and one αNFAT2 sample).
11. Incubate the samples for 4 h at 65 °C and 1300 rpm in a 1.5 mL tube shaker. Add 100 µL of 100% isopropanol, vortex and spin the samples for 5 s at 7000 x g in the small centrifuge.
12. Resuspend the available magnetic beads by thoroughly vortexing, add 10 µL of the beads to every sample and incubate for 1 h at room temperature on a rotating wheel at 6 rpm.
13. After the incubation, spin the tubes for 5 s at 7000 x g in the small centrifuge and place them for 1 min in a magnetic rack. Remove the supernatant by aspiration.
14. Resuspend the beads in 100 µL of wash buffer 1. Invert the tubes to mix and incubate for 5 min at room temperature on a rotating wheel at 6 rpm.
15. Spin the tubes for 5 s at 7000 x g in the small centrifuge, place them for 1 min in a magnetic rack and remove the supernatant by pipetting. Repeat the steps 4.13-4.14 with wash buffer 2.
16. Subsequently, remove the wash buffer by aspiration, resuspend the beads in 55 µL of elution buffer and incubate for 15 min at room temperature on the rotating wheel at 6 rpm to elute the precipitated DNA. Finally, spin the tubes for 5 s at 7000 x g in the small centrifuge, place them for 1 min in a magnetic rack and transfer the supernatant into new 1.5 mL tubes.
    NOTE: Here the protocol can be potentially paused. The eluted DNA should then be stored at -20 °C.

5. Detection of NFAT target genes in CLL cells.

1. Conduct the qRT-PCR reaction in duplicate for every sample as indicated in Table 2.
   NOTE: For the detection of target genes a quantitative real-time PCR (qRT-PCR) is conducted using a commercially available qRT-PCR Mix with a Real-time PCR instrument.
2. Use white 96-well reaction plates for the reactions and the RT-PCR instrument. The cycling conditions are as follows: 95 °C for 30 s, [95 °C for 5 s, 60 °C for 30 s] x 40 cycles.
   NOTE: A serial dilution of the input (undiluted or diluted 1:10, 1:100 and 1:1000 in nuclease-free water) is used to calculate the relative enrichment. In Jurkat cells, IL-2 was used as a positive control³². CD40L, a well-known target of NFAT2 in B cells, was used as a positive control in CLL cells and LCK as an example for a novel target gene of NFAT2 in CLL^33,34. The primers for the CD40L, the IL-2 and LCK promoter region are described in the table of specific reagents and equipment.

6. Normalization and Data Analysis

NOTE: Relative enrichment for the promoter regions of interest (IL-2, CD40L or LCK) is calculated using the IgG-control for normalization.

1. First, determine Ct (threshold cycle) values for the input serial dilution, the IL-2, CD40L and the LCK promoter region via the evaluation software from qRT-PCR data.
2. Define a standard curve for the concentrations via the serial dilution of the input. With this standard curve calculate the concentration for the IL-2, CD40L and the LCK promoter region for each duplicate of every sample.
3. Next, calculate the mean of the duplicates. Then, set the IgG immunoprecipitation sample as a reference. Define the relative enrichment for this sample as 1.0 and compare the other sample (from the NFAT2 immunoprecipitation) to this reference.
4. Finally, calculate the relative enrichment by dividing the concentration of the samples by the concentration of the IgG reference.

Results

Figure 1 shows an exemplary flow cytometry analysis of a CLL patient performed after staining with CD19-FITC and CD5-PE antibodies. Figure 1a shows the gating of the lymphocytes, representing the majority of cells in the blood of CLL patients. Figure 1b shows the proportion of CD19⁺/CD5⁺ CLL cells, which represent 89.03% of lymphocytes in this example. The proportion of CD19

Discussion

The critical steps of performing a successful ChIP assay are the selection of an appropriate antibody and the optimization of the chromatin shearing process²⁵. The selection of the αNFAT2 antibody proved to be particularly challenging during the development of this protocol. While there are several αNFAT2 antibodies commercially available and the majority of these works fine for western blotting and other applications, clone 7A6 was the only antibody which could be successfully used for ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 X PBS	Sigma Aldrich	D8537
1.5 mL tube shaker Themomixer comfort	Eppendorf	5355 000.011	Can be substituted with similar instruments
10X Bolt Sample Reducing Agent	Thermo Scientific	B0009
20X Bolt MES SDS Running Buffer	Thermo Scientific	B0002
37 % Formaldehyde p.a., ACS	Roth	4979.1
4X Bolt LDS Sample Buffer	Thermo Scientific	B0007
Anti-NFAT2 antibody	Alexis	1008505	Clone 7A6
Anti-NFAT2 antibody	Cell Signaling	8032S	Clone D15F1
Anti-NFAT2 antibody ChIP Grade	Abcam	ab2796	Clone 7A6
big Centrifuge	Eppendorf	5804R	Can be substituted with similar instruments
CD19-FITC mouse Anti-human	BD Biosciences	555412	Clone HIB19
CD5-PE mouse Anti-human CD5	BD Biosciences	555353	Clone UCHT2
Density gradient medium Biocoll (Density 1,077 g/ml)	Merck	L 6115
DNA LoBind Tube 1.5 mL	eppendorf	22431021
FBS superior	Merck	S0615
Flow Cytometer	BD Biosciences	FACSCalibur	Can be substituted with similar instruments
Halt Protease and Phosphatase Inhibitor Cocktail (100X)	Thermo Scientific	78440
iBlot 2 Gel Transfer Device	Thermo Scientific	IB21001
iBlot 2 Transfer Stacks, nitrocellulose, regular size	Thermo Scientific	IB23001
iDeal ChIp-seq kit for Histones	Diagenode	C01010059
Ionomycin calcium salt	Sigma Aldrich	I3909
IRDye 680LT Donkey anti-Rabbit IgG (H + L), 0.5 mg	LI-COR Biosciences	926-68023
IRDye 800CW Goat anti-Mouse IgG (H + L), 0.1 mg	LI-COR Biosciences	925-32210
LI-COR Odyssey Infrared Imaging System	LI-COR Biosciences	B446
LightCycler 480 Multiwell Plate 96, white	Roche	4729692001	Can be substituted with other plates in different real-time PCR instruments
Lysing Solution OptiLyse B	Beckman Coulter	IM1400
M220 AFA-grade water	Covaris	520101
M220 Focused-ultrasonicator	Covaris	500295
Magnetic rack, DynaMag-15 Magnet	Thermo Scientific	12301D	Can be substituted with similar instruments
MEM Non-Essential Amino Acids Solution 100X	Thermo Scientific	11140050
Microscope Axiovert 25	Zeiss	451200	Can be substituted with similar instruments
microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm	Covaris	520045
Neubauer improved counting chamber	Karl Hecht GmbH &            Co KG	40442012	Can be substituted with similar instruments
NH4 Heparin Monovette	Sarstedt	02.1064
Nuclease-free water	Promega	P1193
NuPAGE 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well	Thermo Scientific	NP0323BOX
Odyssey® Blocking Buffer (TBS) 500 mL	LI-COR Biosciences	927-50000
Penicillin/Streptomycin 100X	Merck	A2213
PerfeCTa SYBR Green FastMix	Quanta Bio	95072-012
PMA	Sigma Aldrich	P1585
Primer CD40L promotor region forward	Sigma Aldrich		5’-ACTCGGTGTTAGCCAGG-3’
Primer CD40L promotor region reverse	Sigma Aldrich		5’-GGGCTCTTGGGTGCTATTGT -3’
Primer IL-2 promotor region forward	Sigma Aldrich		5’-TCCAAAGAGTCATCAGAAGAG-3’
Primer IL-2 promotor region reverse	Sigma Aldrich		5’-GGCAGGAGTTGAGGTTACTGT-3’
Primer LCK promotor region forward	Sigma Aldrich		5’-CAGGCAAAACAGGCACACAT-3’
Primer LCK promotor region reverse	Sigma Aldrich		5’-CCTCCAGTGACTCTGTTGGC-3’
Rabbit mAb IgG XP Isotype Control	Cell Signaling	# 3900S	Clone DA1E
Real-time PCR instrument	Roche	LightCycler 480	Can be substituted with similar instruments
Roller mixers	Phoenix Instrument	RS-TR 5
RPMI 1640 Medium, GlutaMAX Supplement	Thermo Scientific	61870010
Safety-Multifly-needle 21G	Sarstedt	851638235
SeeBlue Plus2 Pre-stained Protein Standard	Thermo Scientific	LC5925
Shaker Duomax 1030	Heidolph Instruments	543-32205-00	Can be substituted with similar instruments
small Centrifuge	Thermo Scientific	Heraeus Fresco 17	Can be substituted with similar instruments
Sodium Pyruvate	Thermo Scientific	11360070
ß-Mercaptoethanol	Thermo Scientific	21985023
Tris Buffered Saline (TBS-10X)	Cell Signaling	#12498
Trypan Blue solution	Sigma Aldrich	93595-50ML