T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse
- University of California, United States
Figures
Figure 1
Design of novel tdTomato-V5-GCaMP6f fusion probe ‘Salsa6f’ and characterization in living cells.
(A) Several genetically encoded Ca2+ indicators were screened in vitro in HEK 293A cells, by co-transfecting with Orai1/STIM1 and measuring Ca2+ influx after thapsigargin-induced store depletion. …
Figure 2
Generation of a Salsa6f transgenic mouse line targeted to the Rosa26 locus.
(A) Transgenic targeting vector for Salsa6f, inserted between Rosa26 homology arms and electroporated into embryonic stem cells. CAG Pr: cytomegalovirus early enhancer/chicken β-actin promoter; …
Figure 3
Cd4-Salsa6f mice show normal immune cell development and expression.
(A) Experimental design to target expression of Salsa6f in Cd4 cells. (B) Cd4, Cd8 and double-positive cells gated on tdTomato (Salsa6f+ cells) from thymus. (C) Histograms showing percent of Salsa6f+…
Figure 4 with 1 supplement
Functional responses of Cd4-Salsa6f T cells in vitro.
(A) Representative histogram showing cell trace violet (CTV) dilution in Cd4Cre (dark grey), Cd4-Salsa6f (Het) (blue), and Cd4-Salsa6f (Hom) (red) T cells at 72 hr following stimulation with αCd3/28 …
Figure 4—figure supplement 1
Differentiation of Cd4-Salsa6f (Hom) T cells into Th1 and iTregs.
Representative dot plots showing differentiation of Cd4+ T cells from Cd4Cre and Cd4-Salsa6f (Hom) mice into Th1 cells (top panel) and iTregs (bottom panel) after 4 days of stimulation with αCd3/28 …
Figure 5
Single-cell Salsa6f calcium signals in T cells.
(A) Confocal images of Ca2+ signals in activating Cd4+ T cells from Cd4-Salsa6f (Het) mice, after two day stimulation on plate bound αCd3/28 antibody, showing merged green (GCaMP6f) and red …
Figure 6 with 1 supplement
Probe characterization and calibration of [Ca2+] in Salsa6f T cells.
(A) Confocal images of a naive T cell from a Cd4-Salsa6f (Het) mouse. Upper panel: tdTomato (left) and GCaMP6f (right) fluorescence intensity in Ca2+-free Ringer solution. Lower panel: same cell …
Figure 6—figure supplement 1
Comparison of GECI localization in Cd4 T cells from Salsa6f mouse and PC::G5-tdT mouse.
(A,B) Confocal images of Cd4+ T cells purified from a Cd4-Salsa6f (Het) mouse (A) or a Cd4-5GtdT mouse (B), showing merged red and green; cells imaged at the same laser and PMT settings; scale bar = …
Figure 7 with 1 supplement
Ca2+signals in two day activated Cd4+ T cells from Cd4-Salsa6f (Het) mice in response to store-depletion, TCR stimulation and stimulation by Yoda1.
In all panels, average Salsa6f G/R ratios are shown on the left, and representative single-cell traces are shown superimposed on right. Experiments were done in standard Ringer solution (A) or in …
Figure 7—figure supplement 1
Store-operated Ca2+entry and Piezo1 activation in T cells from Cd4-Salsa6f (Het) mice.
Average (left panel) and representative single-cell responses (right panel) to (A) TG-induced SOCE in naive T cells (n = 96 cells), (B) solution exchange with media alone in 2-day activated T-cells …
Figure 8
TCR induced Ca2+ signals in T cell subsets from Cd4-Salsa6f (Het) mice.
Average (left) and representative single-cell Ca2+ traces (right) from confocal time-lapse microscopy showing changes in Salsa6f green/red (G/R) ratio in naive T cells (A), 5-day differentiated Th17 …
Figure 9 with 2 supplements
Lymph nodes from Cd4-Salsa6f (Hom) mice exhibit cell-wide and subcellular Ca2+signals.
(A) Median filtered, maximum intensity projection of a red channel image from a single time point of an explanted lymph node from a Cd4-Salsa6f (Hom) mouse. (B) Green channel image corresponding to A…
Figure 9—figure supplement 1
Imaging lymph nodes of Cd4-Salsa6f homozygous mice.
Cre-mediated expression of Salsa6f in Cd4 T cells reveals endogenous T cell labeling in lymph node. (A) Two-photon images of explanted lymph node from Cd4-Salsa6f (Hom) mouse at various depths …
Figure 9—figure supplement 2
Subtraction of red channel fluorescence improves detection of Salsa6f Ca2+signals.
(A, B) Median filtered, maximum intensity projection of a red channel image from a single time point of an explanted lymph node from a Cd4-Salsa6f (Hom) mouse. Panel A is enlarged and cropped from …
Figure 10
Subcellular Ca2+signals map to different regions of motile, adoptively transferred Cd4-Salsa6f (Hom) T cells.
(A) Histogram of green channel pixel intensities from a representative region of a time-lapse image series of Cd4-Salsa6f (Hom) T cells in a wild-type lymph node after adoptive transfer. Vertical …
Videos
Video 1
Calcium readout of Salsa6f probe in HEK cells.
HEK 293A cells transfected with Salsa6f, first washed with 0 mM Ca2+ followed by 2 μM ionomycin in 2 mM Ca2+; scale bar = 20 µm, time shown in hr:min:s. Images were acquired at 15 s interval and …
Video 2
Single-cell readout of activation in transgenic T cells by Salsa6f.
Cd4 T cells from Cd4-Salsa6f (Het) mice were plated on activating surface coated with anti-Cd3/Cd28. Images were acquired at 5 s interval and played back at 15 frames per second. This video …
Video 3
T cell Ca2+response to Ca2+store depletion by thapsigargin (TG).
Video of maximum intensity projection images of 2-day activated T cells from Cd4-Salsa6f (Het) mouse plated on poly-L-lysine. Scale bar = 20 µm, time shown in hr:min:s. 2 μM TG in Ca2+-free Ringer’s …
Video 4
Activated T cell Ca2+responses to TCR stimulation.
Video of maximum intensity projection images of 2-day activated T cells from Cd4-Salsa6f (Het) mouse plated on anti-Cd3/28-coated coverslip. Scale bar = 20 µm, time shown in hr:min:s. Time interval …
Video 5
T cell Ca2+response to shear and Yoda1.
Video of maximum intensity projection images of 2-day activated T cells from Cd4-Salsa6f (Het) mouse plated on anti-Cd3/28-coated coverslip. Scale bar = 20 µm, time shown in hr:min:s. Time interval …
Video 6
Lymph nodes from Cd4-Salsa6f (Hom) mice exhibit cell-wide and subcellular Ca2+signals.
Time shown in hr:min:s; images were acquired at 5 s intervals. Play back speed = 50 frames per second. Red channel is turned off after beginning to facilitate visualization of green signals. Video …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | Salsa6f | This paper | Fusion of GCaMP6f to tdTomato via a V5 epitope linker (GCaMP6f-V5-tdTomato) | |
| Recombinant DNA reagent | Gt(ROSA)26Sor5’-pCAG- FRT-LSL-Salsa6f-WPRE- bGHpA-AttB-FRT-NeoR- AttP-Gt(ROSA)26Sor3’ cassette | This paper | Salsa6f inserted into a Gt(ROSA)26Sor-pCAG- LSL-(Salsa6f)-WPRE- bGHpA-NeoR cassette. | |
| Transgene (mouse) | Gt(ROSA)26SorpCAG-FRT-LSL- Salsa6f-WPRE-bGHpA-AttB-FRT-NeoR-AttP | This paper | Allele with the above cassette targeted to the ROSA26 locus. | |
| Transgene (mouse) | Gt(ROSA)26SorpCAG-FRT-LSL- Salsa6f-WPRE-bGHpA-AttB/P | This paper | Same as above with Neomycin cassette deleted | |
| Strain, strain background (mouse) | LSL-Salsa6f (F1), LSL-Sals6f (Hom) | This paper | Salsa6f transgene targeted to Rosa26 locus in JM8.N4 mouse embryonic stem (ES) cells. Positive chimeras bred to R26ΦC31o mice to produce LSL- Salsa6f F1 founders and homozygotic LSL- Salsa6f (Hom) mice. See Materials and methods for details. | |
| Strain, strain background (mouse) | Cd4-Salsa6f (Het), Cd4-Salsa6f (Hom) | This paper | LSL-Salsa6f (Hom) mice crossed to Cd4Cre mice to produce heterozygotic and homozygotic Salsa6f- Cd4Cre mice (designated as Cd4- Salsa6f ± and Cd4- Salsa6f+/+ in the paper). | |
| Strain, strain background (mouse) | Cd4Cre mice C57BL/6J | Jackson #017336 | ||
| Strain, strain background (mouse) | C57BL/6J | Jackson #000664 | ||
| Cell line (human) | HEK293A | Invitrogen (#R705-07) | ||
| Transfected construct (synthetic) | Salsa6f | This paper | see above for Salsa6f gene | |
| Transfected construct (synthetic) | G-GECO1, B-GECO1, GCaMP6f, GCaMP6m, GCaMP6s | Addgene | ||
| Antibody | anti-mouse IL-4, IL17A-APC (clone TC11-18H10.1), IFN-Pacific Blue (clone XMG1.2) | BioLegend | ||
| Antibody | Foxp3-PE (clone FJK16s) | ThermoFisher Scientific | ||
| Antibody | αCd3 and αCd28 | Invivogen | ||
| Antibody | αCd3 and αCd28 coated dynabeads | LifeTechnologies Corp. | ||
| Peptide, recombinant protein | recombinant human TGFβ1 | Tonbo Biosciences | ||
| peptide, recombinant protein | recombinant mouse IL-12, IL-23, IL-1β, TGFβ | BioLegend | ||
| Peptide, recombinant protein | recombinant human IL-2 | BioLegend | ||
| Commercial assay or kit | EasySep mouse naïve Cd4 T cell isolation kit | Stem Cell Technologies | ||
| Commercial assay or kit | EasySep mouse Cd4 T cell isolation kit | Stem Cell Technologies | ||
| Chemical compound, drug | Cell trace violet, eFluor 780 | ThermoFisher Scientific | ||
| Chemical compound, drug | Ionomycin, Thapsigargin, Retinoic Acid, PMA | Sigma Aldrich | ||
| Chemical compound, drug | Ghost dye 780 | Biolegend | ||
| Chemical compound, drug | Fura2-AM, Fluo-4 AM | ThermoFisher Scientific | ||
| Software, algorithm | ImageJ/Fiji | NIH | ||
| Software, algorithm | IMARIS | Bitplane | ||
| Other | 35 mm glass chamber | LabTek, ThermoFisher Scientific | ||
| Other | RPMI cell culture medium | Lonza |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32417.024
