Mice
RORceGFP mice were kindly provided by Gérard Eberl (Pasteur Institute, Paris, France) [19]. Ncr1iCre mice were kindly provided by Eric Vivier (CIML, Marseille, France) [20]. Cdh5CreERT2 mice were kindly provided by Ralf Adams (Max Planck Institute for Molecular Biomedicine, Münster, Germany) [21]. Cxcr4flox, Cxcl12flox and RosatdTomato mice were obtained from Jackson Laboratory [22,23,24]. Except for the 3D imaging analysis, 8- to 14-week-old adult male mice were used. All the mice were housed in the specific pathogen-free (SPF) animal facilities and fed with irradiated standard pellet chow and reverse osmosis water. All experiments were performed according to the French ethics committee regulations.
Photothrombotic stroke model
Male mice were anesthetized by intraperitoneal injection of ketamine/xylazine and immobilized on a stereotaxic frame. An incision along the midline from the eye level down to the neck was made and the illuminated area on the exposed skull was determined corresponding to the following coordinates: 2.5 mm posterior to bregma and 2.0 mm lateral to midline. A fiber optic connected to KL 1600LED cold light source was placed on the targeting area after 100 μl of Rose Bengal (10 mg/ml) was injected. Illuminating for 15 min activated the Rose Bengal. After the skin was stitched, animals were put on a heating pad and returned to their home cages until they were fully awake.
Beam-walk sensorimotor test
Mice were trained to walk across a 60-cm-long beam of 0.8 cm diameter, for a minimum of 4 days prior to surgery. For each trial, the mouse was placed at the start and allowed to cross the beam and was given 45 s to reach the goal cage between trials. Each mouse was given five consecutive trials on each test day. The total number of forelimb and hindlimb faults was scored by video recordings. For in vivo depletion of NK cells, mice were injected 100 μg of InVivoPlus anti-mouse Nk1.1 (Clone PK136, Bio X Cell) or InVivoPlus mouse IgG2a (Clone C1.18.4, Bio X Cell) resolved in PBS at P-9 and P-2, respectively.
Administration of 4-hydroxytamoxifen
4-Hydroxytamoxifen (4OHT—Sigma T176) was prepared in 667 μl of ethanol and 6 ml of peanut oil. 160 μl of 4OHT was administered intraperitoneally on 3 consecutive days [25]. Mice underwent 3 weeks of 4OHT before stroke induction.
Flow cytometry
Brains were digested in HBSS containing 0.075 mg/ml liberase TM and 0.2 mg/ml DNase l at 37 ℃ for 60 min. After being harvested and washed by HBSS containing 2% FBS, the brain cells suspended in 30% Percoll were centrifuged at 700 G for 15 min, resuspended in FACS buffer (HBSS containing 2% FBS). The skull was placed in a petri dish containing cold PBS, the edge of which was scored 360° with fine forceps. Then the dura maters were carefully peeled off from the interior side of the skull cap. Dura maters isolated from skull as well as deep cervical, mandibular, axillary and mesenteric lymph nodes were digested in HBSS containing 0.075 mg/ml liberaseTM and 0.2 mg/ml DNase l at 37 ℃ for 20 min. Incubating collected blood with 1 X RBC lysis buffer (Invitrogen, 2139995) for 30 s on ice, then stopping the reaction by HBSS containing 2% FBS. Cell suspensions were centrifuged at 300 G for 7 min and then resuspended in FACS buffer. In the FACS experiments for Fig. 2, Additional file 1: Figs. S1 and S2, cells were blocked in FACS buffer containing 15% normal mouse serum for 15 min and subsequently stained for CD45 (BD biosciences, 564279), CD3 (BD biosciences, 562286), CD8 (BD biosciences, 562283), CD19 (BD biosciences, 562291), Ly6G (BD biosciences, 562700), F4/80 (BD biosciences, 565613), NKp46 (BD biosciences, 562850), CD127 (Ebioscience, 50-1271-80), ST2 (Ebioscience, 12-9333-80), KLRG1 (BD biosciences, 563595), CD4 (BD biosciences, 561099) diluted in FACS buffer for 30 min on ice. The live and dead cells were distinguished using LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life technologies, L23105). Cells were subsequently washed and resuspended in FACS buffer. Cells were blocked in FACS buffer containing 15% normal mouse serum for 15 min and subsequently stained for CD45, CD3e, CD8a, CD19, Ly6G, TCRβ, F4/80, NKp46, NK1.1, CD49a, CD49b, CD127 (Ebioscience, 50-1271-80), ST2, KLRG1 diluted in FACS buffer for 30 min on ice. Cells were washed with FACS buffer and resuspended in FACS buffer containing Sytox Blue. Samples were acquired on the BD LSR Fortessa-X20 cytometer and data were analyzed using FlowJo (version 10, LLC) software.
Immunofluorescence
The mice were perfused with PBS containing 5U/ml of Heparin followed by 4% paraformaldehyde (PFA). The brains were extracted and fixed by 4% PFA overnight at 4 ℃. 50-μm Vibratome sections were cut and blocked in EBT buffer (EBSS with 1% BSA and 2% Triton-X100) containing 10% serum for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies diluted in EBT buffer containing 3% serum for 24 h at 4 ℃. After washing 3 times with PBS containing 0.2% Triton-X100, the sections were incubated with fluorochrome-coupled secondary antibodies diluted in EBT buffer containing 3% serum for 12 h at 4 ℃. After washing 3 times with PBS, the sections were cleared in Histodenz medium (4 g Histodenz (Sigma D2158) in 3 ml of 0.02 M phosphate buffer) for 24 h at room temperature and then mounted in Histodenz medium. Confocal images were acquired on a Zeiss-LSM880 confocal microscope and processed by ImageJ.
Nissl staining
Vibratome sections were cut and mounted on positive charged plus slides (ThermoFisher scientific, SuperFrost Plus). The slides were placed in 1:1 alcohol/chloroform overnight and then rehydrated through 100% and 95% alcohol. Then the slides were placed into 0.1% cresyl violet for 10 min and rinsed quickly in distilled water. The sections were cleared with xylene for 10 min after being differentiated in 95% alcohol for 5 min and dehydrated in 100% alcohol for 10 min. For quantification of the infarct volumes, the infarct area was measured by using ImageJ and the total volume was calculated as the sum of infarct areas multiplied by the section interval.
Whole mount immunofluorescence
The RORceGFP mice were perfused with PBS containing 5U/ml of heparin followed by 0.4% paraformaldehyde (PFA). The brains were harvested and fixed by 0.4% PFA overnight at 4 ℃ and subsequently placed in permeabilization solution for 3 days. Subsequently, they were incubated in blocking medium PBS-MT (1% skim milk, 0.4% Triton X-100, 5% NDS and 5% NGS) for 3 days and subsequently incubated with anti-GFP (AVES, GFP-1020), anti-CD3 (BD biosciences, 563024), anti-KLRG1 (BD biosciences, 563595) and anti-NKp46 (BD biosciences, 562850) in PBS-MT with 3% NDS plus NGS for 10 days. After washing with PBS containing 0.2% Triton X-100, brains were incubated with donkey anti-chicken Cy3 (Jackson Immunoresearch, 703-166-155), goat anti-Armenian hamster 647 (Jackson Immunoresearch, 127-605-160), goat anti-Syrian hamster 488 and donkey anti-rat 790 (Jackson Immunoresearch, 712-655-153) in PBS-MT with 3% NDS as well as NGS for 10 days. Brains were washed with PBS and dehydrated by methanol series (20%, 40%, 60%, 80% 1 h each and 100% 2 h). Subsequently, the brains were placed in 66% dicholoromethane (Sigma, 270997), 33% methanol overnight, and afterwards in 100% dicholoromethane for 15 min. The brains were cleared in BABB (2:1 benzyl alcohol (Honeywell, 108006)/benzyl benzoate (Acros organics, 105862500)) overnight. Brains were acquired using the light sheet Ultramicroscope version II (LaVision BioTec). 3D imaging analysis were performed using Imaris software (Version 9.1.0, Bitplane).
Quantitative PCR
Total RNA was prepared by TRIzol reagent (Sigma). cDNA synthesis was carried out using RevertAid RT Kit (ThermoFisher scientific). The transcripts were amplified on the 7500 Fast Real-Time PCR System. The amplification of Cxcr4 and the corresponding Gapdh was detected by Taqman probes (Cxcr4-Mm01292123_m1, Gapdh-Mm99999915_g1; ThermoFisher scientific), combining with Taqman Fast Advanced Master Mix (ThermoFisher scientific). The amplification of Cxcl12 and the corresponding Gapdh was detected by the following primers: Cxcl12_forward 5’-tcaagcatctgaaaatcctcaaca-3’, Cxcl12_reverse 5’-ttcgggtcaatgcacacttgt-3’, Gapdh_forward 5’-tgtgtccgtcgtggatctga-3’, Gapdh_reverse 5’-cctgcttcaccaccttcttga-3’, combining with TB Green Premix Ex Taq (Takara). Relative expression was calculated by ∆∆CT method.
Statistical analysis
Graphs in all figures were analyzed using GraphPad Prism (v.7.03) software. Statistical significance of differences between groups in Figs. 2B, 3E, 4E, 5C and E were analyzed by two-way ANOVA, and in Figs. 4F and 5D were compared using t-test. All the cartoons were generated using BioRender.
Staining panels for FACS
Staining panel I for FACS in Fig. 2, Additional file 1: Figs. S1 and S2.
CD45
|
BV395
|
BD biosciences, 564279
|
CD3
|
PE-CF594
|
BD biosciences, 562286
|
CD8
|
PE-CF594
|
BD biosciences, 562283
|
CD19
|
PE-CF594
|
BD biosciences, 562291
|
Ly6G
|
PE-CF594
|
BD biosciences, 562700
|
F4/80
|
PE-CF594
|
BD biosciences, 565613
|
NKp46
|
BV421
|
BD biosciences, 562850
|
CD127
|
eFluor 660
|
Ebioscience, 50-1271-80
|
ST2
|
PE
|
Ebioscience, 12-9333-80
|
KLRG1
|
PerCP-Cy5.5
|
BD biosciences, 563595
|
CD4
|
PE-Cy7
|
BD biosciences, 561099
|
RORγt
|
RORγt-eGFP
|
Sparwasser et al., 2004
|
ReporterLive/Dead
|
Fixable Blue Dead Cell Stain Kit
|
Life technologies, L23105
|
Staining panel II for FACS in Figs. 3, 4, 5, Additional file 1: Figs.S4, S6 and S7.
CD45
|
BV395
|
BD biosciences, 564279
|
CD3
|
BV510
|
BD biosciences, 563024
|
CD8
|
V500
|
BD biosciences, 560776
|
CD19
|
BV510
|
BD biosciences, 562956
|
Ly6G
|
BV510
|
Biolegend, BLE127633
|
F4/80
|
BV510
|
BD biosciences, 743280
|
TCRβ
|
BV510
|
BD biosciences, 563221
|
NKp46
|
BV421
|
BD biosciences, 562850
|
NK1.1
|
Alexa Fluor 700
|
Ebioscience, 56-5971-82
|
CD49a
|
BV711
|
BD biosciences, 564863
|
CD49b
|
PE-Cy7
|
Miltenyi, 130-116-370
|
CD127
|
eFluor 660
|
Ebioscience, 50-1271-80
|
ST2
|
PE
|
Ebioscience, 12-9333-80
|
KLRG1
|
PerCP-Cy5.5
|
BD biosciences, 563595
|
Live/Dead
|
Sytox Blue
|
Life technologies, S34857
|