Apc rat antimouse cd4

The cell apoptosis state of MCF‐7 cells on scaffolds was carried out by an Annexin V‐FITC/PI assay kit (Neobioscience, China). The cells were collected and stained with Annexin V‐FITC and PI in binding buffer. For evaluating HIF‐1α expression, cells were treated by 2% formaldehyde, 0.1% Triton X‐100, and anti‐HIF‐1α (Cell signaling technology, USA) for 30 min away from light. For immune cells analysis, a single cell suspension (100 µL, 5×10⁷ cells mL⁻¹) of dissociated tumor tissue was seeded on the scaffolds and cultured for 48 and 72 h. The cancer cells were harvested, rinsed with PBS, and resuspended. Then, the collected cells with Fixable viability stain 780, PerCP‐Cy5.5 Rat Antimouse CD45, FITC Hamster Antimouse CD3e, APC Rat Antimouse CD4, and PE Rat Antimouse CD8a (BD Biosciences, USA) were incubated at 4 °C for 30 min away from light. The tests were carried out on a FACS Celesta flow cytometer (BD Biosciences, USA) and calculated the data with FlowJo 10.

For flow cytometry-based sorting of infected cells, mice were infected with 10⁴ PFU MHV68-H2bYFP, a phenotypically wild-type virus that expresses eYFP under control of the H2b promoter [44 (link)]. At 16 dpi, splenocytes were prepared and blocked as described above. Cells were then stained with APC rat anti-mouse CD4 at 1:200 (BD Biosciences, 553051), APC rat anti-mouse CD8α at 1:200 (BD Biosciences, 553035), APC rat anti-mouse CD14 at 1:100 (BD Biosciences, 560634), and APC-Cy7 rat anti-mouse CD19 at 1:200 (BD Biosciences, 557655). Infected B cells (CD4^-CD8^-CD14^-CD19⁺YFP⁺) and non-infected B cells (CD4^-CD8^-CD14^-CD19⁺YFP^-) were sorted using a BD FACSAria II flow cytometer (BD Biosciences). Sorted cells were immediately subjected to RNA extraction using an RNAqueous-Micro kit (Ambion, AM1931) prior to qRT-PCR analyses.

Draining lymph nodes were dissected from mice with psoriatic-like inflammation or control mice. Subsequently, the cells were dissociated and collected via centrifugation for 5 min at 1500 rpm. The cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 500 ng/mL ionomycin (Sigma-Aldrich). Thereafter, GolgiPlug (BD Biosciences) and GolgiStop (BD Biosciences) were added, and the cells were incubated for an additional 3 h (total incubation time 4 h) and stained for surface markers, followed by fixation and permeabilization with Cytofix/Cytoperm solution (BD Pharmingen). The cells were stained to detect IL-17 A and subjected to FACS analysis on Canto2 (BD Biosciences). The following antibodies were used in this study: Live/Dead Fixable Aqua Dead cell stain kit (Invitrogen, L34957, Carlsbad, CA), PerCP Hamster anti-mouse CD3 (BD Pharmingen, 553,067, RRID: AB_394599, San Diego, CA), APC Rat anti-mouse CD4 (BD Pharmingen, 553,051, RRID: AB_398528), FITC Hamster anti-mouse γδTCR (BD Pharmingen, 553,177, RRID: AB_394688), PE Rat anti-mouse CD8 (BD Pharmingen, 553,032, RRID: AB_394571), and PE-Cy7 Rat anti-mouse/rat IL-17 A PE/Cy7 (eBioscience, 25-7177-82, RRID: AB_10732356, San Diego, CA).