Conjugate formation and processing for ultrastructural studies

Conjugate formation between CTL expressing APEX-constructs and EL4 targets, and cytochemical processing for TEM ultrastructural studies 

Note. This protocol refers to the preparation of conjugates formed between SIINFEKL-presenting EL4 cells and activated CTL prepared from Tg(TcraTcrb)1100Mjb Rag1^{tm1Bal/tm1Bal} (referred to as OTI) mice bred on a C57BL/6 background, nucleofected with APEX-expressing plasmids 4-6 days after activation and 16-24h prior to use. Please find details of preparation of activated CTL from OT1 spleens, EL4 cells, plasmids and nucleofection procedures in the original paper [DOI: 10.1126/science.abp8933].

CELLS AND REAGENTS

Cells

1. Activated CTL prepared from OT1 mice spleens (Stinchcombe et al., 2023). Maintained in culture in RPMI 1640 medium (Sigma-Aldrich, # 1640) with 10% FBS (LabTech, # FBS-SA), 50 mM β-mercaptoethanol (Thermo Fisher Scientific, # 31350010), 10 U/ml recombinant murine IL-2 (Peprotech, # 212-12), 2 mM L-Glutamine (Sigma-Aldrich, # G7513-100ML), 1 mM sodium pyruvate (ThermoFisher Scientific, # 11360070) and 50 U/ml penicillin and streptomycin (Sigma-Aldrich, # P0781-100ML). 

Used for conjugation studies 16-24 hr after nucleofection with constructs expressing APEX-tagged probes (i.e. 5-8 days after activation).

2. EL4 cells (Ritter et al., 2015): Maintained in DMEM (Sigma-Aldrich, # D5030-10X1L) supplemented with 10% FBS and 2 mM L-glutamine.

Reagents 

1. Hemin: (Sigma-Aldrich, # 9030) – use at 7 μg/ml final concentration

2. SIINFEKL activating peptide: (Cambridge Bioscience) 

3. RPMI 1640 medium: (Sigma-Aldrich, # 1640) 

4. Paraformaldehyde: 16% aqueous solution (Electron Microscopy Sciences, # 15710)

5. Gluteraldehyde: 25% aqueous solution (Agar scientific, # R1020)

6. Sodium cacodylate: (Agar Scientific, # R1102)

7. Trizma base: (Sigma-Aldrich, # T1503) 

8. HCl: (BDH, # 101254H)

9. 3,3'-diaminobenzidine tetrahydrochloride hydrate (DAB): (Fluka 32750-25G-F)

10. 30% H₂O₂ solution: (Sigma, # H1009)

11. Osmium tetroxide: 0.2% aqueous solution (TAAB, # O007) 

12. Urynal acetate (UA): (TAAB, # U007)

SOLUTIONS AND STOCK SOLUTIONS

1. Haem solution for pre-incubation of CTL expressing APEX constructs 

Prepare a concentrated solution (e.g. 5-10x) of hemin in full CTL culture medium just before use:

• Measure sufficient hemin powder to give a final concentration of 7 μg/ml in the final CTL culture.
• Add the hemin to the required volume of fresh complete CTL culture medium and vortex.
• Filter through a 0.45 μm syringe filter to sterilise the solution and use soon after preparation.

2.  SIINFEKL peptide stock solution 

• Prepare a sterile stock solution of 0.1mM in RPMI or dH₂O, and store at -20°C.
• Use at 1:100 for conjugation experiments.
   

3. PBS - at RT and pre-cooled to 4°C

4. Fixative solutions - Prepare and use in fume hood

- 2x fixative solution: 3% glutaraldehyde + 4% paraformaldehyde in PBS 

- 1x fixative solution: 1.5% glutaraldehyde + 2% paraformaldehyde in PBS 

For CTL, especially conjugates, fixative temperature at use should be at least RT and ideally 37°C (incubate briefly immediately prior to use to avoid denaturation).

5. 0.1 M sodium cacodylate, pH 7.4 - pre-cool to 4°C

• Dissolve 4.28 g of sodium cacodylate powder in 200 ml dH₂O for 0.1 M solution.

Check pH and adjust as required.

NOTE: Sodium cacodylate is an organic arsenic compound and should be handled with care.

6. Tris buffer (TB): 50 mM Tris-HCl, pH 7.6

Stock solutions: - Store at RT

• 0.2 M Tris: dissolve 12.1 g Trizma base in 500 ml dH₂O
• 0.1 N HCl: add 4.4 ml HCl to 500 ml dH₂O (prepare in fume hood).

Tris buffer (TB): - Prepare fresh and precool to 4°C before use

• 25 ml 0.2 M Tris stock solution + 40 ml 0.1 M HCl stock solution + 35 ml 4°C dH₂O

Check pH before use and adjust if required. If the pH is incorrect the enzyme will not be active and will not give a reaction product.

7. DAB reaction solution: 0.75 mg/ml DAB, 0.021% (v/v) hydrogen peroxide in TB

DAB stock solution:

• Prepare 40x concentrated (i.e. 30mg/ml) DAB solution in TB.

Store in aliquots at -20°C protected from light. Thaw immediately before use.

DAB reaction buffer: 

Prepare immediately before use as follows:

• Add 250 μl of DAB stock solution to 20 ml 4°C TB and mix.
• Add 20 μl of 30% w/w H₂O₂, shake rapidly to mix and immediately add to samples.

Note: this solution will discolour and go off quickly if not used immediately, and/or exposed to light during a reaction. Therefore, use immediately, use in the dark and discard any residual solution.

8. dH₂O - pre-cooled to 4°C

9. 0.1% Osmium tetroxide - prepare and handle in fume hood. 

NOTE Osmium is a heavy metal and should be used with great care and both solid and liquid waste disposed of appropriately. Refer to local/institute COSH details, guidance and regulations before use.

• Prepare 0.1% solution by diluting 0.2% osmium tetroxide stock 1:1 immediately before use in clean dH₂O pre-cooled to 4°C.

Ultraclean, dust free equipment must be used (e.g. individually wrapped pipettes) to avoid unwanted osmium precipitation.

10. 2% Uranyl acetate (UA) - prepare and use in fume hood. 

NOTE:  UA is a regulated substance and should be used, and all waste disposed of, with care and under local/institute guidance and regulations.

• Prepare 2% solution in dH₂O (e.g. 1 g in 50 ml) at least 24 hr in advance to allow the powder to dissolve completely.
• Mix using a vortex.

Store in sealed tube at 4°C in dark (e.g. sealed with parafilm and wrapped in foil).

EXPERIMENTAL PROCEDURE 

Ensure all solutions are placed at the correct temperature prior to starting (i.e. media and fixative solutions are warmed; post-fixation processing solutions are at 4°C).

1. Cell preparation

1. Before starting 

Calculate the total number of OT1 CTL and EL4 target cells required for the experiment, given:

• Samples are plated at final total cell concentrations of 1-2x10⁶ cells/ml.
• For conjugation, CTL and EL4 targets are mixed 1:1.
• 0.5 ml cell solution is used per sample for a 4-well plate.

2. 14-22 hours before experiment (~2 hr after nucleofection)

Pre-incubate CTL expressing APEX constructs with haem:

• Place the required volume of nucleofected CTL in a separate flask or plate.
• Calculate the amount of hemin solution needed to give a final concentration of 7 μM (see solution preparation above) and add the filter-sterilised concentrated hemin solution directly to the CTL sample.
• Incubate at 37°C for 14-22 hours.

3. 40-60 min before experiment

Pre-incubate EL4 cells with activating peptide:

• Place the required amount of EL4 targets in a 15 ml tube and add 1μM SIINFEKL peptide (1:100 dilution of 0.1 mM stock solution).
• Incubate at 37°C for 40-60 min.

2. Cell conjugation and plating

1. Wash unbound ova (EL4), hemin (OT1) and serum from OT1 and targets

• Take the pre-incubated EL4 (in 15 ml tube) and CTL cell populations from 37°C, and transfer the hemin-incubated OT1 CTL to a 15 ml tube.
• Fill each tube with RPMI lacking serum (RPMI-), and centrifuge (200 g, 5 min, >22°C).
• Resuspend cell pellets in RPMI- and recentrifuge (200 g, 5 min, >22°C) 1-2x more.
• Resuspend each final pellet in RPMI- to give final solutions of 1-2x10⁶ cells/ml.
• If required, remove CTL to be analysed unconjugated and put in a separate tube.

2. Conjugation of OT1 CTL to EL4 targets and cell plating

For conjugation: 

• Mix EL4 cell solution 1:1 with OT1 CTL population (t=0).
• Leave in tube on bench for 2-5 min.
• Pipette the suspension up and down gently and plate 0.5 ml aliquots into individual wells of Nunclon 4-well multi-dishes (Thermofisher # 176740).
• Place at 37°C to allow cell adhesion, conjugation and killing.
• Fix at 20, 40 or 60 min from cell mixing (t=0), depending on stage of cell-cell interaction of interest.

For unconjugated CTL: 

• Plate cell suspension directly into 4-well multi-dishes at 0.5 ml/well and place at 37°C to allow cell adhesion.
• For cells plated as control cells for conjugation experiment, fix at the times as the conjugated cells, otherwise fix once cells are adhered to the plate.

Note: Plate samples to be fixed at different time points (and/or different subsequent processing steps in distinct 4-well multi-well dishes) so that the processing of early time point samples does not interfere with the longer incubation of later time point samples. 

3. Fixation and DAB cytochemical reaction

1. Fixation

• Take samples from 37°C and place in a fume hood at RT.
• Add 0.5 ml pre-warmed 2x fixative solution to the medium directly above each sample (giving a final concentration = 2.5% glutaraldehyde/1.5% paraformaldehyde/well).
• Leave in fume hood at RT for 10 min.
• Exchange fixative for 1 ml fresh 1x fixative solution and leave at RT for further 10 min.
• Transfer samples to ice/4°C and leave for 10-30 min at 4°C.

Retain samples at 4°C and precool all solutions for all subsequent steps up to dehydration to minimise diffusion of the cytochemical reaction product.

2. Washing (4°C)

• Remove final fixative solution with ~6-8x rinses/well with 4°C PBS. Once the fixative is removed, the samples can be moved from the fume hood.
• Wash each well of each sample using the following washing procedure:

- 16x quick rinses (4x (4x rinses/well)) with 4°C PBS then leave in final rinse for 5 min.

- 16x quick rinses as above with 4°C 0.1 M sodium cacodylate, pH 7.4, and leave 5 min.

- Repeat 16x rinse with 4°C 0.1 M sodium cacodylate, pH 7.4, and leave 5 min.

3. DAB cytochemical reaction (4°C)

• Rinse each well >3x with 4°C 50 mM Tris-HCl, pH 7.6 (Tris buffer, TB) to remove the 0.1 M sodium cacodylate solution and equilibrate the samples before the DAB reaction.
• Mix components of DAB reaction solution to give a final solution of 0.75 mg/ml DAB, 0.021% (v/v) hydrogen peroxide in TB.
• Add immediately to samples at ~1 ml/well and incubate at 4°C in the dark for 15-30 min, depending on the APEX-construct.
• Terminate the reaction by quickly removing the reaction buffer and rinsing >2x with TB.

Note: For constructs with abundant and/or concentrated APEX-tagged protein expression, the DAB-reaction product may be visible in the cells by light microscopy as a brown reaction product. These reactions can therefore be monitored using a normal TC microscope. Low level or dispersed proteins may not be visible by light microscopy and the success of the reaction product in these cases is only assayable at the TEM level.

• Rinse the samples 16x in 4°C 0.1 M sodium cacodylate, pH 7.4, and leave 5 min, as above.
• Repeat this washing step a further 1-2 times.
• Rinse the samples a couple of times with 4°C dH₂O, then return samples to fume hood.

4. Post-fixation and staining with osmium and uranyl acetate (UA)

Please see notes on safe handling of osmium and UA 

1. Osmium incubation (4°C)

• In fume hood, replace final rinse solution with 4°C 0.1% aqueous osmium tetroxide, using ultra clean pipettes, at < 0.5 ml/well (ensuring cells are completely covered in liquid).
• Clean the lids of the plates well to remove residual solution, place them over the plates and leave for 50-60 min in dark (e.g. covered with foil).
• Remove osmium solution and rinse quickly 8x with dH₂O, disposing of waste solution as specified by local regulations.
• Wash the samples 16x with 4°C dH₂O and leave 5 min as above.
• Repeat this washing step with 4°C dH₂O a further 2 times. Do not use sodium cacodylate as this will precipitate UA.

2. UA incubation (4°C)

• Replace final rinse solution with 4°C 0.5% aqueous UA solution at > 0.5 ml/well (ensuring cells are completely covered).
• Clean lids of plates thoroughly, replace them over samples, seal plates with parafilm and leave overnight in dark (e.g. covered with foil) at 4°C. Samples can be removed from the fume hood and placed in 4°C rooms if plates are sealed.
• Remove UA and rinse quickly 8x with dH₂O, disposing of waste solution as specified by local regulations.
• Wash the samples 16x with 4°C dH₂O and leave 5 min, as above.
• Repeat this washing step with 4°C dH₂O a further 2x.

5. Dehydration, embedding and analysis 

Following UA incubation and washing, samples can be transferred from ice/4°C and processed using standard dehydration and embedding protocols, then sectioned and analysed by TEM.

• For thin section TEM, cut 50-150 nm sections using a microtome.
• For tomography, serial 250-300 nm sections are collected.

NOTES

1. Prolonged exposure to fixatives inactivates enzymes including peroxidases. Samples therefore should not be stored in fixative before the DAB reaction and, for best results, samples should be carried straight through from fixation to the UA incubation on the same day.

2. Many reagents used in EM preparation are dangerous and need to be handled and disposed of appropriately. Please consult local safety, COSH, and waste disposal forms and regulations, and use in appropriate environments with appropriate protection.

3. For untransfected CTL (i.e. not expressing APEX constructs), the DAB-reaction step is omitted and samples can remain at RT after fixation. The UA step can be carried out at either 4°C using 0.5% solution overnight, as described above or RT using 2% solution for 2 hours.

References

A. T. Ritter, Y. Asano, J. C. Stinchcombe, N. M. G. Dieckmann, B.-C. Chen, C. Gawden-Bone, S. van Engelenburg, W. Legant, L. Gao, M. W. Davidson, E. Betzig, J. Lippincott-Schwartz, G. M. Griffiths, Actin depletion initiates events leading to granule secretion at the immunological synapse. Immunity 42, 864-876 (2015).

J. C. Stinchcombe, Y. Asano, C. J. G. Kaufman, K. Böhlig, C. J. Peddie, L. M. Collinson, A. Nadler, G. M. Griffiths, Ectocytosis renders T cell receptor signaling self-limiting at the immune synapse. Science 380, 818-823 (2023).