Confocor 3

Time-lapse LSM images for ccRICS analysis were acquired by a combination system consisting of an LSM510 and a ConfoCor3 (Zeiss, Jena, Germany) with a temperature control system. ATTO488 was excited by a 488-nm Ar-ion laser and ATTO647N was excited by a 633-nm He–Ne laser through a water-immersion objective (C-Apochromat, 40×, 1.2 NA; Zeiss). Two lasers simultaneously illuminated the observation volume at the same position. Emission signals were collected by photon counting at 505–610 nm for ATTO488 (green channel) and >650 nm for ATTO647N (red channel) with avalanche photodiodes (APDs). The pixel size was 22 nm, which is 5–10 times smaller than the focused laser spot, and the pixel dwell time was 5–50 μs per pixel.

For detection of IL-23R, cells were cultured with 1:100 diluted goat anti-IL-23R (Abcam) at 4° C and then with 1:100 diluted rabbit anti-goat PE-conjugated secondary antibody (ProteinTech Group, Wuhan, China) at 4° C in the dark. After that, half cells were analyzed by flow cytometer and half cells were further stained with hochest33342 (5 μg/ml, Beyotime) for 10min followed by observation under the fluorescence microscope (LSM 710 and ConfoCor 3, Zeiss, Germany).
For detection of CD133, saponin permeabilized cells were incubated with PE labeled anti-CD133 (Miltenyi Biotec, Germany) and then were analyzed by FACS Calibur.

To elucidate the interacting carrier molecule in the serum, we first incubated Cy3-labelled ASO or Toc-HDO (100 pmol) for 20 min at 37 °C with mouse HDL fraction (99 μl) (‘ex vivo' samples). Next, we intravenously injected 3 mg kg⁻¹ Cy3-labelled ASO or Toc-HDO to the mouse, and corrected serum 10 min after injection (‘in vivo' samples). The measurements were performed using the ConfoCor 3 module in combination with the LSM 510 (Carl Zeiss MicroImaging, Göttingen, Germany) equipped with the C-Apochromat × 40/1.2 W objective. A HeNe laser (543 nm) was used for Cy3-labelled ASO and Toc-HDO excitation in PBS or serum, and excitation of the Nile Red (Tokyo Chemical Industry, Tokyo, Japan)-labelled HDL or LDL fraction. Emission was filtered through a 560- to 615-nm band-pass filter. Samples were placed into an 8-well Lab-Tek chambered slide (Nalgene Nunc International, Rochester, NY), and diffusion time was measured at room temperature. Autocorrelation curves obtained from 10 measurements with a sampling time of 20 s were fitted with the ConfoCor 3 software package to determine the diffusion time of samples.

FCS and FCCS was carried using either a Zeiss LSM780 or Zeiss 710 with Confocor 3 mounted on an AxioObserver Z1 microscope with a 63x C-apochromat, 1.2 NA water-immersion objective. Zen 2010B software was used for data collection and analysis. EGFP fluorescence was excited with 488nm laser light and emission collected between 500 and 530 nm. DsRed-express was excited with 561nm laser light and emission collected between 580 and 630 nm. The protocols as outlined in Kim et al. (Kim et al., 2007 (link)) were followed, with 10 x 10 s runs used for each measurement. FCS was used to quantify the total number of fluorescent molecules per cell as previously described (Bagnall et al., 2015 (link)). The confocal volume had previously been estimated at 0.59 ± 11 fL (mean ± SD) using Rhodamine 6G of known diffusion rate, and WT HeLa cells in suspension were imaged by confocal microscopy to give volume estimates of 1420 ± 490 fL and 6110 ± 3580 fL for nucleus and cytoplasm respectively. (For FCCS controls see Appendix Section E).