Imaging Cytosolic Calcium in T Lymphocytes: a protocol for use with genetically encoded or chemical Ca2+ indicators

Amit Jairaman1 and Michael D. Cahalan1,2,* 

Affiliations:

¹ Department of Physiology & Biophysics, University of California, Irvine, CA 92697-4561, USA.

² Institute for Immunology, University of California, Irvine, CA 92697, USA.

• Corresponding author: Michael D. Cahalan, Email: mcahalan@uci.edu

One Sentence Summary: We describe a detailed protocol for calcium imaging of T cells in vitro that can be applied to genetically encoded or chemical indicators in a wide variety of cell types.

Introduction

Calcium indicators have revealed second-messenger cytosolic Ca²⁺ signals leading to cellular activation sequelae for nearly 40 years since the pioneering work of Roger Tsien. Tsien developed dyes based on the Ca²⁺ chelator EGTA, including quin-2 (Rink et al., 1982), and improved chemical indicators such as the excitation ratiometric indicator fura-2, the emission ratiometric indicator indo- 1 (Grynkiewicz et al., 1985), and several others including combinations of fura-red and fluo-4 that can be implemented for ratiometric imaging (McQuade et al., 2020). Chemical Ca²⁺ indicators are typically loaded into cells using a permeable ester-linked parent compound that is cleaved by intracellular esterases. To overcome issues of dye loading and leakage, a variety of approaches have been taken to develop genetically encoded Ca²⁺ indicators based on mutated GFP-like structures, including FRET probes such as the Cameleons (Miyawaki et al., 1997), and tandem probes such as Salsa6f (Dong et al., 2017b) with red TdTomato (TdT) linked through an epitope tag to the green GCaMP6f Ca²⁺ indicator. This simple combination imparts powerful capabilities: tracking cells through the red TdT signal in the absence of Ca²⁺ elevations when the GCaMP6f fluorescence is very dim; ratiometric imaging to eliminate motility artifacts; convenient single-wavelength (920 nm) femtosecond excitation for two-photon microscopy. Other advantages include: excellent dynamic range with Ca²⁺ sensitivity in cytosol from 100-1000 nM; stable expression restricted to cytosol in unperturbed mouse T cells; no effect on T cell development and activation; and ability to calibrate. Implemented in a transgenic mouse line for Cre-dependent expression, Salsa6f enables Ca²⁺ signaling to be monitored in a wide variety of cell types. Our group has used Salsa6f to examine Ca²⁺ signaling by confocal microscopy in isolated T cells from Cd4-Salsa6f mice (Dong et al., 2017b), macrophages from LSL-Salsa6f-Vav1^Cre/+ mice (Atcha et al., in press), and astrocytes from GFAP-Cre Salsa6f mice (Wakida et al., 2020); and by two photon imaging of T cells in lymph nodes (Dong et al., 2017a) and pathogenic Th17 cells in spinal cord (Othy et al., 2020).

This set of protocols describes the preparation of T lymphocytes for in vitro calcium imaging experiments. It is applicable to lymphocytes and to developing thymocytes that are resident in the thymus. Mature T and B lymphocytes migrate in the circulation throughout the body and home into lymphoid organs where they search for antigen. Lymphocytes can be easily isolated and purified from blood or lymphoid tissue, and are maintained as suspension cultures or in wells coated with antibodies against CD3 and CD28. The methods described below using either genetically encoded or chemical Ca2+ indicators can also be readily adapted to adherent cells. Footnotes provide additional details.

The Day before T cell Isolation:

1）Preparing antibody-coated 6-well plates: Coat 6-well plate with antibodies against mouse CD3 (anti-CD3) and CD28 (anti-CD28). Dissolve the antibodies in PBS at final concentration of 2.5 mg/ml in a 15 ml falcon tube. Use 2 ml of PBS/ well (12 ml for all 6 wells). Mix the antibody solution by inverting the tube several times and let stand for a few minutes before plating 2 ml/ well. Seal the 6-well plate using parafilm to prevent evaporation of the antibody solution and incubate the 6-well plate at 4 oC overnight¹. This substrate will be used to activate CD4+ T cells isolated from mouse lymph node (LN) and spleen.

2）Preparing antibody-coated imaging chambers: If planning to image TCR-activated Ca2+ signals in freshly isolated T cells on plate-bound anti-CD3/anti-CD28 the next day, prepare imaging chambers by coating 35 mm glass-bottom dishes (LabTek chambers, 1.5 coverglass) with anti-CD3/anti-CD28. Use 2.5 mg/ml of each antibody in PBS and add 250 ml per 35 mm chamber². Incubate at 4 oC overnight. Make sure the chamber is on a flat surface to ensure uniform concentration of the coated antibody throughout.

3）Preparing Poly-L-Lysine (PLL) coated chambers³: Add 250 ml of 100% PLL (1 mg/ml in mol. grade water) to the center of 35 mm glass-bottom dishes at room temperature (RT). Let it sit for 1 hour. Wash off the PLL 2-3 times with PBS and let the chambers dry. They are now ready to be used anytime within 2-3 weeks. Store at room temperature (RT) away from light.

4）Prepare mouse T cell media containing RPMI with 10% FCS, 2 mM L-glutamine, 1x non-essential amino acids, 1 mM sodium pyruvate, 55 µM β-mercaptoethanol, 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml of Amphotericin B.

On the Day of T cell Isolation:

5）Dissect LN and spleen from mouse. Place in 3 ml of RPMI complete or T cell media. Use the bottom of 3ml syringe to mash and pass cells through a 70 mm cell strainer into a 50 ml falcon tube. Spin cells down and re-suspend in EasySep (PBS + 0.1% FBS + 0.5 mM EDTA) or MACS buffer (PBS + 0.1% BSA +0.5mM EDTA) depending on the kit being used to isolate CD4⁺ cells. Follow kit instructions to isolate CD4⁺ T cells.

6）After isolation is complete, wash cells by spinning them down for 5 min at 1000 RPM with excess media to remove all traces of EDTA. Resuspend cells in 4-5 ml of fresh warmed T cell media and count the cells. Each mouse yields approximately 10 million CD4⁺ T cells. Meanwhile, bring the 6-well plate to RT in the TC Hood and wash the antibody-coated wells 2-3 times with PBS to remove the uncoated antibody. Add approximately 2 million cells into each 6 well to activate T cells. Cells should be suspended in T cell media + 30 U/ml of murine interleukin-2 (mIL-2). Add 4 ml of media per well⁴.

Preparation of Cells for Imaging⁵:

7）Cells need to be re-suspended at the right concentration of T cell media to yield an appropriate density for imaging. When working with freshly isolated T cells (which are small), re-suspend at 4-5 million/ml. This gives a 60-70% confluency when plated on anti-CD3/anti-CD28 coated or PLL coated 35 mm chambers. When working with 2-day activated T cells, which are larger in size, re-suspend at 1 million/ ml. At the above concentrations, cells should be kept at 37 oC (either in 1.5 ml centrifuge tube or 15 ml falcon tube, depending on the volume) in the TC incubator on the day of imaging⁶. For imaging, plate approximately 50-100 ml⁷ of cell-suspension into the central well of the 35 mm chamber, and place the imaging chamber (and remaining cells) back in the incubator. Incubation time required for cell adhesion is: 15 min for PLL⁸ and approximately 1 hr for anti-CD3/anti-CD28⁹.

8）After cell attachment, wash cells two times by gently adding 2 ml of the final imaging solution to the side of the 35 mm chamber (and not directly on the cells) to remove unattached cells. Bathe cells in the imaging buffer and mount on the microscope for imaging.

9）To synchronize plating and imaging of T cells on PLL and anti-CD3/28: When doing multiple chambers, it is sometimes necessary and always helpful for reproducibility to ensure the same plating times between different samples. Plan accordingly. Illustrated below is an example of a protocol that could be followed for imaging on PLL (for 4 imaging chambers). Each box is a 15 min interval.

Loading cells with Calcium indicator dyes (Fluo-4 AM plus Fura-Red AM or Fura-2 AM for Ratiometric Imaging)

10）Prepare stocks of the Ca²⁺ dyes at 2 mM in DMSO. Stocks are stored at -20°C. Bring stock to RT before imaging. All dyes are used at 3 mM final concentration. Dye will be mixed with equal volume of pluronic acid to facilitate loading into cells. Pluronic acid is stored at RT and needs to warmed up to 40 oC for about 20-30 min for it to liquefy (it is a gel at RT which will make pipetting small volumes difficult).

11）Preparing a working stock: Prepare dye solution in T cell media at 2x the concentration that will be used finally. Each 35 mm chamber requires 100 ml of cell-suspension (50mL of 2x Dye solution + 50 ml of 2x cell suspension). As an example, to image 6 chambers, prepare at least 300 ml of dye at 6 mM (1.5 ml of 2mM stock in 500 ml of medium). Add

1.5 ml of dye (for Fluo-4 and Fura-red imaging, add 1.5 ml of each dye) to a 1.5 ml tube, then add equal volume of pre-warmed pluronic acid and mix vigorously by vortexing several times. Finally add the required volume of T cell media (500 ml) and vortex again. This is the ‘intermediate’ 2x stock of the dye.

12）To label cells for plating on PLL: For each imaging chamber, take 50 ml of 2x dye solution and mix with 50 ml of 2x cell-suspension¹¹ in a 1.5 ml tube. Incubate for 15 min at 37 oC, then add to the center of a PLL coated dish. Plate for 15 min. Wash 2-3 times with imaging buffer and image on the scope.

13）The 2x dye solution and 2x cell suspension need to be stored in the TC incubator for further experiments. Illustrated below is a typical protocol for dye labeling-plating- imaging on PLL for 6 different chambers (Each box represents a 15 min interval: Blue box- loaded cells in 1.5 ml tube, Red box- dye-loaded cells plated on PLL; Green box- wash the dye off the cells, mount and image):

14）For experiments on anti-CD3/anti-CD28: For each imaging chamber, take 50 ml of 2x dye solution and mix with 50 ml of 2x cell-suspension in a 1.5 ml tube. Plate immediately to the center of an anti-CD3/ 28 coated dish. Plate for 30 min. Wash 2-3 times with imaging buffer before imaging.^12-13.

Footnotes:

1. As a general rule, plating antibodies at 4°C overnight leads to better coating. If imaging chambers need to be prepared in a hurry on the same day as imaging, 2-3 hr plating at 37°C  can also lead to decent antibody coating.
2. Add 250 ml of the antibody solution to the central 14 mm well of the 35 mm MatTek chamber (Cat# P35G-1.5-14-C). Concentration of coating antibodies can be varied depending on the research question being asked. Lower concentrations of antibodies may be used to study Ca²⁺ signals in response to smaller CD3 activation stimulus. Cross- linking of CD3 is the primary stimulus for Ca²⁺ signaling. Cross-linking of CD28 provides co-stimulatory signal which may not be critical for short-term Ca²⁺ signaling but  helps the T cells to attach to the substrate better and may be important to sustain Ca²⁺ signaling after several hours of plating.
3. PLL-coated chambers provide positively charged, but non-activating substrates to attach T cells and are useful to study changes in cytosolic Ca²⁺ in response to receptor activators and inhibitors, Ca²⁺ channel agonists and antagonists etc. These are usually added by perfusion/ solution exchange to the 35 mm dishes containing the cells.

4. Cells activated for 2 days are ideal for imaging, although cells can be used freshly after isolation (resting T cells) or after 1, 2 or 3 days of activation. Note that some cells may start losing their viability after 48 hr due to excessive activation. Spinning the cells at a low speed (~700 RPM) may help to get rid of some dead cells, but not all.

5. Note that the plating protocol under this section applies to T cells that express genetically encoded Ca2+ probe and do not require loading with chemical Ca²⁺ dyes. When using dyes, additional loading steps need to be followed (see Section on Dye loading)
6. Ensure sufficient CO₂ exchange by keeping the cap loose or open in the incubator. Gently mix cells occasionally (~every 30 min) to prevent formation of cell pellets. Cells will be fine at RT for short periods of time but it is important to have them at 37°C for imaging, especially with anti-CD3/anti-CD28 coated dishes. It is highly recommended to plate and incubate cells for imaging in a TC Hood close to the microscope. Move cells very gently to the microscope. Moving imaging chambers that contain T cells plated on anti- CD3/anti-CD28 over longer distance can easily cause detachment of the cells. Cells plated on PLL are a little more forgiving, and tend to attach better.
7. Note that having a low volume of cell suspension (50-100 ml) in the imaging dish for up to an hour should not adversely affect cell viability. But additional media should be gently added to the sides of the chamber if cells are to be kept in the incubator for a longer time.
8. Plating times for cells on PLL: Do not exceed 15 min. Cells are not very happy after 30- 40 min on PLL. This gives ~15-20 min of imaging time. Less than 10 min of plating may lead to poor cell attachment.
9. Plating times for cells on anti-CD3/anti-CD28: While 1 hr plating is more than sufficient to ensure proper cell attachment and TCR signaling activation, shorter and longer plating times are also possible. Cells can potentially attach within 5-10 min, which might be useful to study the early Ca²⁺ events of TCR signaling. One may also consider adding cells to an imaging chamber already mounted on the microscope to view very early events of T cell signaling. For this, cells would need to be added close to the imaging field right where the objective is, at a very high density and low volume (for example 500,000 cells in ~10 ml). Cells can be imaged up to 24-48 hr after plating as well, as long as the cells are healthy and bathed in sufficient fresh media. Freshly isolated cells are more amenable to imaging after 1-2 days, as 2 day-activated cells are more likely to die due to over stimulation of TCR.
10. 1 mM Ca²⁺ Ringer solution contains (in mM): 155 NaCl, 4.5 KCl, 1 CaCl₂, 0.5 MgCl₂, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). Ca²⁺-free Ringer solution contains (in mM): 155 NaCl, 4.5 KCl, 1.5 MgCl₂, 10 glucose, 1 EGTA, 10 HEPES (pH adjusted to 7.4 with NaOH).
11. Note that the cell-suspension will also need to be at 2x concentration in this case. Mixing with the dye solution at 1:1 ratio will result in the correct concentration for both dye and cells. So, use an adjusted concentration of 10 million/ml for resting or freshly isolated T cells and 2 million/ml for activated T cells.
12. Note that on both PLL and anti-CD3/28, total dye incubation time with cells is 30 min.
13. For anti-CD3/28 coated chamber, longer dye incubation times may be used if cells do not attach to the substrate in 30 min. Consider reducing dye concentration in that case. Make sure the loading conditions are consistent between all samples. This is critical for proper comparison, especially for Fluo-4 and Fura-Red imaging.

KEY RESOURCES TABLE

References

Dong, T.X., S. Othy, M.L. Greenberg, A. Jairaman, C. Akunwafo, S. Leverrier, Y. Yu, I. Parker, J.L. Dynes, and M.D. Cahalan. 2017a. Intermittent Ca²⁺ signals mediated by Orai1 regulate basal T cell motility. Elife. 6.

Dong, T.X., S. Othy, A. Jairaman, J. Skupsky, A. Zavala, I. Parker, J.L. Dynes, and M.D. Cahalan. 2017b. T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse. Elife. 6.

Grynkiewicz, G., M. Poenie, and R.Y. Tsien. 1985. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 260:3440-3450.

McQuade, A., Y.J. Kang, J. Hasselmann, A. Jairaman, A. Sotelo, M. Coburn, S.K. Shabestari, J.P. Chadarevian, G. Fote, C.H. Tu, E. Danhash, J. Silva, E. Martinez, C. Cotman, G.A. Prieto, L.M.

Thompson, J.S. Steffan, I. Smith, H. Davtyan, M. Cahalan, H. Cho, and M. Blurton-Jones. 2020. Gene expression and functional deficits underlie TREM2-knockout microglia responses in human models of Alzheimer's disease. Nat Commun. 11:5370.

Miyawaki, A., J. Llopis, R. Heim, J.M. McCaffery, J.A. Adams, M. Ikura, and R.Y. Tsien. 1997.

Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin. Nature. 388:882-887.

Othy, S., A. Jairaman, J.L. Dynes, T.X. Dong, C. Tune, A.V. Yeromin, A. Zavala, C. Akunwafo, F. Chen, I. Parker, and M.D. Cahalan. 2020. Regulatory T cells suppress Th17 cell Ca²⁺ signaling in the spinal cord during murine autoimmune neuroinflammation. Proceedings of the National Academy of Sciences of the United States of America. 117:20088-20099.

Rink, T.J., S.W. Smith, and R.Y. Tsien. 1982. Cytoplasmic free Ca2+ in human platelets: Ca²⁺ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett. 148:21-26.

Wakida, N.M., V. Gomez-Godinez, H. Li, J. Nguyen, E.K. Kim, J.L. Dynes, S. Othy, A.L. Lau, P. Ding, L. Shi, C. Carmona, L.M. Thompson, M.D. Cahalan, and M.W. Berns. 2020. Calcium Dynamics in Astrocytes During Cell Injury. Front Bioeng Biotechnol. 8:912.

In Press:

Atcha, H., A. Jairaman, J. R. Holt, V. S. Meli, R. R. Nagalla, P. K. Veerasubramanian, K. T. Brumm, H. E. Lim, S. Othy, M. D. Cahalan, M. M. Pathak, and W. F. Liu. Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing. Nat Commun. (in press).