Elyra system s 1 microscope

BHK-21 cells at 70% confluence were transfected with 1 µg pCI::AtHAP2::GFPnes, pCI::AtHAP2::H2B-RFP, pCI::PfHAP2::GFPnes, pCI::PfHAP2::H2B-RFP, pCI::PfHAP2p::GFPnes and pCI::PfHAP2p::H2B-RFP respectively. Control cells were transfected with pCI::GFPnes or pCI::H2B-RFP (vectors). Four hours after transfection, the cells were washed 4 times with DMEM with 10% serum (Invitrogen), 4 times with PBS, and detached using Trypsin (Biological Industries). The transfected cells were collected in Eppendorf tubes, resuspended in DMEM 10% FBS, and counted. Equal amounts of H2B-RFP and GFPnes cells were mixed and seeded in glass-bottom plates (12-well black, glass-bottom #1.5H; Cellvis) and incubated at 37 °C in 5% CO₂. 18 h after mixing 20 µM FdUrd was added to the plates and 24 h later, the cells were fixed with 4% PFA in PBS. Nuclei were stained with 1 µg/mL DAPI. Micrographs were obtained using wide-field illumination using an ELYRA system S.1 microscope and a Plan-Apochromat 20X NA 0.8 lens (Zeiss); images were recorded with a iXon + EMCCD camera (Andor). The GFP + RFP mixing index was calculated as the number of nuclei in mixed cells, GFPnes cytoplasm with red (H2B-RFP) and blue (DAPI) nuclei out of the total number of nuclei of fluorescent cells in contact and expressed as percentage.

BHK-21 cells grown on 24-well tissue-culture plates with glass bottom or coverslips, were transfected when they reached 70% confluence using 1 µg pCI::PfHAP2::H2B-RFP or pCI:: PfHAP2p::H2B-RFP. 18 h post-transfection, 20 µM 5-fluoro-2ʹ-deoxyuridine (FdUrd) was added to the plates to block the cell cycle and 24 h later, the cells were fixed with 4% paraformaldehyde (PFA; EM grade, Bar Naor, Israel) in PBS, followed by incubation in 40 mM NH4Cl to block free aldehydes, permeabilized in 0.1% Triton X-100 and blocked in 1% Fetal Bovine Serum (FBS). We stained the cells with anti-V5 mAb (Invitrogen, 1:500), the secondary antibody was donkey anti–mouse coupled to Alexa Fluor 488 (Invitrogen, 1:500) and supplemented with 1 µg/mL DAPI [21 (link)]. Micrographs were obtained using wide-field illumination using an ELYRA system S.1 microscope and a Plan-Apochromat 63X NA 1.4 objective (Zeiss); images were recorded with an iXon + EMCCD camera (Andor).