Amg evos fl digital inverted microscope

I. scapularis (ISE6), D. andersoni (DAE100), and A. americanum (AAE2) embryonic derived cell lines were obtained from Dr. Ulrike Munderloh at the University of Minnesota through a material transfer agreement. Tick cell lines were maintained in Cellstart^® 25 cm² flasks (Greiner bio-one), containing L15C300 medium supplemented with 5% FBS, 5% TPB, and 0.1% LPC kept at 34 °C^{84 (link)}. Bacterial infection experiments and subcultures were performed in whole 25 cm² flasks (Greiner bio-one) when cells reached confluency, as evaluated by light microscopy with an AMG EVOS Fl digital inverted microscope (Thermo Fisher Scientific). HL-60 (CCL-240) cells were obtained from ATCC and grown in RPMI-1640 medium with l-Glutamine (Quality Biological) supplemented with 10% FBS (Gemini Bio-Products) and 1% GlutaMax^TM (Gibco). DH82 cells were a kind gift from Jere McBride at University of Texas Medical Branch. These cells were grown in DMEM/F12 (1:1; Gibco) supplemented with 10% FBS (Gemini Bio-Products). Both cell lines were maintained at 37 °C and 5% CO₂. Cells numbers were determined using a TC20™ automated cell counter (Bio-Rad).

Cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 for 15 min at RT. Fixed cells were blocked with 2% bovine serum albumin (BSA) (Sigma-Aldrich) for 4 h at RT and then incubated overnight with the primary antibodies (Table 1) at 4°C. The following day, cells were washed thrice with dPBS and blocked again for 1 hour with 2% BSA at RT. Secondary antibodies, such as goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor 555 (Abcam), mouse anti-goat Alexa Fluor 555 (Abcam), goat anti-chicken Alexa Fluor 488 (Abcam), and goat anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific), at 1:500 were added for 1 hour at RT. Cells were triple-washed with dPBS, and nuclei were stained using DAPI (Invitrogen) at 1:10,000 diluted in a 1x dPBS solution for 5 min. Cells were washed once after the DAPI addition with dPBS and mounted using the ProLong Gold Antifade Reagent (Invitrogen). Images were acquired using the AMG EVOS FL digital inverted microscope (Thermo Fisher Scientific.) and the LEICA DMi8 wide-field microscope (Leica Microsystems).