Hoechst

Adherent cells cultured in chamber slides were serum-deprived, then stained with 100 ng/mL Hoechst (BD Biosciences, Le Pont de Claix, France) during 30 min, stimulated 15 min with 1 μg/mL fluorescent CSF-1 before wash and living cell observation with a spinning disk microscope (Andor Technology, Belfast, UK) during 10 min (1-min delay between each image).

The siRNAs for Stat3, Jak2 (pools of three specific 19-25 nt siRNAs) and control siRNA were purchased from Santa Cruz. The Constitutive active Stat3-C plasmid was a kind gift from Professor Zhijie Chang (Tsinghua University) [49 (link)]. Icaritin was purified from Epimedium at the Beijing Shenogen pharma group with over 99 % purity. Cisplatin and AG490 were obtained from Sigma Aldrich. Sorafenib was purchased from Selleck. U0126 was purchased from Cell Signaling Technology and Hoechst was from BD Pharmingen. Human recombinant IL-6 was from Peprotech and S3I-201 was purchased from Santa Cruz Biotechnology.

Calcein AM (BIOTIUM, USA) was used to stain all the MCF-7 cells, and Hoechst (Life, USA) was used to stain the DNA in all the cell nuclei. Labeling was completed by two approaches: one, by putting the staining reagents into the assay directly, and the other by putting into the cell suspension. Next, 1 mL of 1× PBS containing 10 mg mL⁻¹ (1%) bovine serum albumin (BSA, Solarbio, China) and 0.05% Tween-20 was used with cancer cells to reduce the non-specific cell adhesion on the surface of the structure. For cells captured on the Hoechst chip, Cytokeratin-FITC (BD Biosciences) and CD45-PE (BD Biosciences) were used for on-chip staining. A small limited number of unlabeled tumor cells were spiked into the lysed and whole blood. Those artificial patient bloods were processed through the chip followed by washing with PBS, fixing, and permeabilization. Anti-cytokeratin (BD Biosciences) and anti-CD45 (BD Biosciences) and Hoechst applied in 1% bovine serum albumin were utilized for all the samples. After washing, then the samples were ready for microscopic imaging.^{45 (link)}

Frozen tissue (~25 mg) from either frontal cortex or cerebellar hemisphere (see table of demographics) was dissected at −20 and subjected to dounce homogenization followed by sucrose gradient centrifugation as previously described. Tissue was primarily BA10 or lateral cerebellar hemisphere although some samples were named only as frontal cortex or cerebellum. Nuclei were filtered and incubated for 5 min in 1:1,000 Hoechst (Invitrogen H3569, Waltham MA). A total of 10,000 Hoechst + nuclei from the suspension were then sorted directly into 10× Genomics RT buffer (Pleasanton, CA) on a chilled plate holder to remove doublets, debris, and dying nuclei on a FACS Aria (BD Biosciences, Franklin Lakes, NJ) with a low-pressure nozzle (SI Appendix, Fig. S2). Following sorting, reverse transcriptase enzyme was added on ice, and nuclei were immediately processed for encapsulation in the 10× Chromium controller. cDNA and libraries were prepared according to the 10× documentation protocol for 3′ gene expression v3 chemistry.

To study the interaction of swollen, opsonized and non-opsonized conidia with CRs on DCs, cells were incubated with conidia at a 1:2 ratio. Confocal slides were coated with Poly-L-Lysine (Sigma-Aldrich) for 2h at 37°C. DCs were stained with Hoechst, anti-human CD11b- or CD11c-Alexa594 and anti-human DC-SIGN Alexa633 (all from BD) and added to the PBS washed slides for at least 1h until they were adherent. FITC-labeled opsonized and non-opsonized conidia of the different strains were added to the cells for 6h to have comparable conditions as during binding/internalization experiments evaluated by flow cytometry. Pre-warmed D-PBS was used to remove unbound conidia and slides were fixed using 1% formaldehyde for 20min. Slides were mounted onto coverslips with Mowiol (Sigma) and used for microscopy. Images were acquired on a Leica SP5 (x63 Glycerin) confocal microscope at the Biooptics core facility, CCB, Innsbruck, Austria. Deconvolution was performed using Huygens Professional and images were merged using ImageJ Software.

On the day of analysis, cells were pelleted by 500× g at 4 °C and then washed twice with PBS. For 4′,6-diamidino-2-phenylindole (DAPI)/Ki67 staining, cells were resuspended in 100 µL PBS, and 10 µL Ki67-FITC (Abcam, Cambridge, MA, USA) antibody was added for every 1 million cells. After 30 min incubation at room temperature in the dark, cells were washed with PBS then DAPI (1 µg/mL, Sigma-Aldrich, Darmstadt, Germany) was added. Cells were then incubated at room temperature for 15 min. In order to quantify the staining, 355/450 nm excitation/emission was used for DAPI, 488/530 nm excitation/emission was used for Ki67-FITC. The flow cytometry analysis was performed on a BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA, USA). The Ki67-negative population threshold was determined based on a DAPI-only staining control. Data were analyzed using FACSDiVa analysis software (BD Biosciences, San Jose, CA, USA).
Similarly, the RNA levels were determined using pyronin (4 µg/mL, Acros Organics, Geel, Belgium) and Hoechst (10 µg/mL, BD Biosciences, San Jose, CA, USA) staining. Cells were treated with a mixture of both stains for 30 min in the dark at room temperature, and then underwent flow cytometry analysis. The 355/450 nm excitation/emission was used for Hoechest, while 488/582 nm excitation/emission was used for pyronin.