Buffer t

For siRNA manipulation of monocytes, immediately after isolation from PBMC samples, monocytes were washed in PBS, centrifuged at 300 × g for 5 min, supernatant discarded and the cell pellet resuspended at 3 × 10⁷ cells/mL in buffer T (ThermoFisher). A NeonTM tube was filled with 3 mL of buffer E2 and inserted into the NeonTM Pipette Station. HIF1Α, HIF2A, JUN or Silencer™ negative control siRNA (ThermoFisher) were added to the isolated monocyte/buffer T mixture so that the final working concentration of siRNA was 100 nM. The siRNA/monocyte/buffer T mixture was taken up into a 100 µL NeonTM Pipette tip (ThermoFisher) and electroporated with the Neon transfection system (ThermoFisher) using the settings: 1920 V, 25 ms, 1 pulse. The electroporated cells were either shared across 2 wells of a 24-well plate with 1 ml of antibiotic free media (CTL medium + 10% FCS) for LD culture; or placed in a single well of a 96-well plate with 100 µl antibiotic-free media for HD culture. Electroporated cells were incubated in antibiotic-free CTL medium for 48 h before FcγR expression was analyzed by flow cytometry.

HAP1 cells (2 × 10⁷ cells) were collected and resuspended in 0.2 ml of OPTI-MEM (Life Technologies). Cells were mixed with 10 μg of plasmids and electroporated once at 250 V for 20 ms using ECM830 (BTX). Alternatively, cells and 10 μg of plasmids were mixed and resuspended in 100 μl of buffer T (Life Technologies) and electroporated at 2000 V for 10 ms twice using Neon (Life Technologies).

To evaluate the role of NKEF in the response of RBCs to VHSV, RBCs were transfected with siNKEF or siGFP (187 pmol of siNKEF or siGFP per 0.5 × 10⁶ cells) resuspended in Buffer T (Life Technologies). At 24 h post-transfection, RBCs were exposed to VHSV MOI 1 at 14 °C. In the case of RTG-2 cells, to evaluate the role of NKEF in the response to VHSV, they were transfected with siNKEF or siGFP (100 pmol of siNKEF or siGFP per 1.23 × 10⁵ cells) resuspended in Buffer R (Life Technologies). At 48 h post-transfection, cells were exposed to VHSV MOI 0.1 (RTG-2) at 14 °C. In both cases, at 3 hpe, medium was refreshed with RPMI 2% FBS and incubated for 24 h. Then, cells were resuspended in TRK lysis buffer for RNA extraction and kept at −80 °C.