Application of dual Nr4a1-GFP Nr4a3-Tocky reporter mice to study T cell receptor signaling by flow cytometry

Summary This protocol uses Nr4a1-GFP Nr4a3-Tocky mice to study T cell receptor (TCR) signaling using flow cytometry. It identifies the optimal mouse transgenic status and fluorochromes compatible with the dual reporter. This protocol has applications in TCR signaling, and we outline how to obtain high-quality datasets. It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al., 2020.


SUMMARY
This protocol uses Nr4a1-GFP Nr4a3-Tocky mice to study T cell receptor (TCR) signaling using flow cytometry. It identifies the optimal mouse transgenic status and fluorochromes compatible with the dual reporter. This protocol has applications in TCR signaling, and we outline how to obtain high-quality datasets. It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al., 2020.
CRITICAL: Use of heterozygous Nr4a3-Tocky in combination with Nr4a1-GFP leads to reduced expression of Nr4a3-Timer (Jennings et al., 2020). Therefore, it is strongly recommended that Nr4a3-Tocky homozygous mice are used when these are mated to Nr4a1-GFP mice.
Note: The following primers are made up to 100 mM final concentration as master stocks. From these 1 in 10 dilutions of working 10 mM stocks are made.
Note: Timer BAC F+R produce a PCR product of approximately 180 bp; Il2ra F+R product is approximately 200 bp. Note: Depending on machine and SYBR green master mix, the DCt may vary. Controls with known zygosity should be used to identify DCt ranges. i. Figure 1 shows typical DCt using this protocol and ABI 7900HT machine. j. Determine zygosity of line and use Nr4a3-Tocky homozygous in combination with Nr4a1-GFP for experiment.

Panel design
Timing: 3-4 h CRITICAL: Key to high-quality analysis is optimal panel design and this should be validated before undertaking experiments. Here we illustrate the compensation matrix and optimized fluorochrome combinations using a BD LSR Fortessa X-20 machine. It is by no means exhaustive but represents a good starting point for panel design. The assumption is that Timer-Blue and Timer-Red do not spectrally overlap (Subach et al., 2009), and therefore no compensation is required between these two channels (Bending et al., 2018b). Single color controls can be generated through using a Nr4a1-GFP line (or equivalent GFP expressing cell line), and Nr4a3-Tocky cells. To generate a single Blue and Red color control: a. Place 1 million Nr4a3-Tocky splenocytes into a 96 well round bottom plate containing 10% FBS RPMI (v/v) and stimulate with 5 mg/mL soluble anti-CD3 for 4 h.
Note: Nr4a3-Blue expression will remain for long as as a TCR stimulus is applied in vitro; however, the transition of Blue to Red has a half-life of approximately 4 h, meaning stimulus for longer periods will increase Red fluorescence of cells. b. Harvest cells and maintain on ice until analysis on flow cytometer, gate on Nr4a3-Blue + (BV421 channel) and Nr4a3-Red À (PE-Texas Red channel) cells to use as a single color Blue control. c. Use unstimulated Nr4a3-Tocky T cells and gate on Nr4a3-Blue À Nr4a3-Red + to generate a single Red color control.
Note: Figure 2 shows that Blue, Red and GFP colors show no overlap (see also Note: Caution should be used when using BV711 and BV605, and PE-Cy7 due to the spectral overlaps between them and Timer-Red and Timer-Blue channels. We recommend that single color compensation controls for all tandem dyes use the dye that is in the final panel. We advise not using PE or BV510 due to the close proximity to Nr4a3-Red and Nr4a3-Blue. FITC is not compatible with GFP. Compensation matrix generated on FlowJo software indicating percentage spectral overlap between various channels. Single color controls were generated using activated splenocytes from pure Nr4a3-Tocky mice (for Timer-Blue and Timer-Red; Figure 2), or thymus from OTI Nr4a1-GFP Nr4a3-Tocky (for Nr4a1-GFP single color; Figure 2). Non-transgenic cells were stained with the following antibodies to generate single color controls: anti-mouse CD5 APC; viability dye eFluor780 (APC-Cy7); anti-mouse TCRbeta PerCP-Cy5.5; anti-mouse CD8 BUV737; anti-mouse CD4 BUV395; anti-mouse CD8 PE-Cy7; anti-mouse-CX3CR1 BV605; anti-mouse CD11c BV711; anti-mouse CD103 PE.

MATERIALS AND EQUIPMENT Mice
All animal experiments were performed in accordance with local Animal Welfare and Ethical Review Body at the University of Birmingham and under the authority of a Home Office project license, P18A892E0A held by D.B. Animals were housed in specific pathogen-free conditions with appropriate housing conditions and husbandry as specified by NC3Rs. Nr4a3-Tocky mice expressing a BAC containing FT Fast mCherry mutant (Subach et al., 2009) under the influence of Nr4a3 regulatory regions on the C57BL/6J background as previously described (Bending et al., 2018a;Bending et al., 2018b) were bred to Nr4a1/Nur77-GFP mice expressing a BAC containing GFP transgene under the influence of Nr4a1 regulatory regions on C57BL/6J background as previously described (Moran et al., 2011). These were mated to OTI mice (expressing the TCR (Va2, Vb5) of the OVA257-264-specific CTL clone 149.42) to generate OTI Nr4a3-Tocky Nr4a1-GFP mice.
Flow cytometer -BD LSR FORTESSA X-20 Data were acquired on a BD LSR FORTESSA X-20 flow cytometer, with the configuration detailed below. Dyes require careful compensation considerations are marked with an asterisk. Other cytometers with similar setups would also be suitable. Alternatives: Alternative fluorochrome formats are available for many channels, however these should be tested in advance for compatibility to Nr4a3-Blue, Nr4a3-Red, and Nr4a1-GFP. Alternative flow cytometer settings and filters are also available, but each user should optimize voltages and panel design to their own machines.

STEP-BY-STEP METHOD DETAILS
Assessing T cell receptor signal duration in the regulation of Nr4a1 and Nr4a3 expression using Nr4a1-GFP and Nr4a3-Tocky dual reporter mice Generation of single-cell splenocyte suspension

Timing: 1-2 h
This part generates a single-cell suspension of splenocytes for use in experimental cultures.

Harvest spleen from
Nr4a1-GFP Nr4a3-Tocky mice (Jennings et al., 2020) in TC hood. a. Mice are euthanized by cervical dislocation, and death confirmed by cessation of circulation. b. Spleen is removed, ensuring complete detachment of the pancreas and placed into a 1.5 mL Eppendorf containing 0.5 mL 10% FBS RPMI. 2. Generation of single-cell suspension in tissue culture hood. a. Into a sterile 5 mL petri dish, place a 70 mm cell strainer and add the spleen and 1 mL of 10% FBS RPMI buffer. (Alternatively, a strainer can be placed in a 50 mL Falcon tube for processing). b. Using the syringe plunger from a 5 mL syringe, gently force the spleen through the cell strainer. c. Using a P1000 pipette add 1 mL 10% FBS RPMI buffer to wash the strainer. d. Filter the splenocyte solution back through the strainer by taking from suspension in the petri dish and pipetting back onto the strainer. Repeat this 8-10 times using P1000 pipette until suspension is homogenous. e. Pool the splenocyte suspension by transferring it into a 15 mL Falcon tube. f. Wash strainer with 1 mL 10% FBS RMPI and transfer this to the 15 mL Falcon tube. g. Centrifuge Falcon tube at 500 3 g for 5 min at 15 C-20 C. h. Decant supernatant and proceed to perform red blood cell lysis by resuspending the cell pellet in 1 mL of RBC lysis buffer. i. Incubate for 2 min on ice. j. Top up the 15 mL Falcon tube with 9 mL 10% FBS RPMI and centrifuge at 500 3 g for 5 min at 15 C-20 C. k. Decant supernatant and resuspend cell pellet at cell concentration of 5 million/mL in 10% FBS RPMI and keep cells on ice.

Culture setup
Timing: 0-4 h (B) Splenocytes from Nr4a3-Tocky (pure) line were analyzed for Nr4a1-GFP versus Nr4a3-Red (left) or stimulated for 3 h with anti-CD3 and then Nr4a3-Blue + Nr4a3-Red À cells analyzed for Nr4a1-GFP versus Nr4a3-Blue. These single color controls were used to make the compensation matrix in Table 1 This part established how to modulate the TCR signal length in splenocyte cultures from Nr4a1-GFP Nr4a3-Tocky mice.
Optional: Cultures are set up on 96 well round bottom plates (Corning) in a final volume of 190 mL, with 10 mL volume used to add the PP2 inhibitor, however depending on cell numbers and desired interactions other plate formats may also be suitable.
Note: PP2 is reconstituted in sterile DMSO to a master stock concentration of 20 mM.
Note: During incubation, the staining mix can be prepared (see below).

12.
After 240 min proceed immediately to staining.

Staining for flow cytometric analysis
Timing: 0.5-1 h 13. Centrifuge culture plate at 500 3 g for 3 min, flick plate to decant supernatant and while still inverted blot against paper towel. 14. Add 25 mL of staining mix prepared as below in 2% FBS PBS (v/v). In our hands viability dye staining can be performed simultaneously with surface staining without adversely affecting performance.
Note: Include single color compensation controls in addition to GFP, Timer-Blue, and Timer-Red.  16. Add 180 mL of wash buffer to each well and centrifuge plate at 500 3 g for 3 min, flick plate to decant supernatant and while still inverted blot against paper towel. 17. Resuspend pellet in 150 mL 2% FBS PBS and transfer to 5 mL FACS tube (BD Biosciences). Wash well with 150 mL 2% FBS PBS and transfer to FACS tube to make 300 mL final volume. 18. Place on ice in the dark and acquire samples on the flow cytometer within 2 h of staining.

Stain Stain dilution
CRITICAL: Cells should be analyzed as soon as possible following staining. In our hands, slow maturation of Blue to Red can occur after prolonged periods (e.g., overnight) at 4 C.

EXPECTED OUTCOMES
Using this protocol, it is expected to see an early increase in Nr4a1-GFP expression commencing within as little as 5-10 min of stimulation time. Troubleshooting 2Nr4a3-Blue expression will start to increase after 30-60 min of stimulation. After 12 min of TCR signaling 50% of T cells should be Nr4a1-GFP + . After 70 min of TCR stimulation 50% of T cells should be Nr4a3-Blue + . With 4 h of TCR signaling >85%-90% of CD4 + and CD8 + T cells should be Nr4a1-GFP + Nr4a3-Blue + (see Figure 4D in (Jennings et al., 2020)) Troubleshooting 4

LIMITATIONS
This protocol is designed for the analysis of Nr4a1-GFP and Nr4a3-Timer expression patterns by flow cytometry. Other fluorescence-based analysis techniques such as confocal microscopy have not been tested or validated on the dual reporter. This protocol also relies on the analysis occurring within 2 h of staining, and samples being kept on ice in the dark to prevent the maturation of Timer-Blue into Timer-Red. This protocol is also not compatible with fixation and intranuclear factor staining with kits such as the eBioscience Foxp3 / Transcription Factor Staining Buffer Set.

TROUBLESHOOTING
Problem 1 A diagonal population of Blue + Red + cells appear in unstimulated controls.

Potential solution
This is typically seen in preparations from tissues that have increased autofluorescence of some cells.
To reduce background, we advise having a ''dump'' channel open such as the PerCP-Cy5.5 channel.
If no dye is used in this channel, background noise is reduced substantially (Figure 3).

Problem 2
Weak signal in Blue channel but strong signal in GFP after stimulation. Potential solution This is typically caused by using Nr4a3-Tocky heterozygous BAC mice in combination with Nr4a1-GFP BAC mice. While Nr4a3-Tocky heterozygous mice are usable alone, when crossed with Nr4a1-GFP BAC mice the Nr4a3-Timer signal is quenched. We advise checking zygosity of line and repeating experiment with a confirmed Nr4a3-Tocky homozygote with Nr4a1-GFP heterozygote.

Problem 3
Ex vivo Timer-Red signal is dim for single color staining control.

Potential solution
Splenocytes can be activated with soluble anti-CD3 for 4 h before washing and keeping in fridge for 24 h. The Blue proteins will mature to the terminal Red form and provide a brighter single Red color control.

Problem 4
Cells show a clear Blue + Red + pattern, even after a short 4-h stimulation.

Potential solution
This may occur if cells are not analyzed immediately. The half-life of the timer protein is 4 h, and this process can be stopped by keeping cells on ice before acquisition. We strongly recommend analysis within 2 h of staining of samples. Overnight incubation in the fridge is likely to reduce Timer-Blue and increase Timer-Red signal.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, David Bending (d.a.bending@bham.ac.uk ).

Materials availability
Nr4a3-Tocky mice are available under MTA from Dr Masahiro Ono, (Imperial College London, UK). Nr4a1/Nur77-GFP mice are available from the Jax lab (strain number 016617). (C) The result is that lymphocytes from wild-type mice gated as PerCP-Cy5.5 À display very low noise in the Timer-Blue versus Timer-Red channels.