Clear bottom 96 well plate

Activities of enzymes contained within the lysosomes (cathepsin-D), mitochondria (citrate synthase), or peroxisomes (catalase) were measured from the Percoll gradient fractions dissolved in 0.1% Triton X-100 (final). Cathepsin-D activity was assayed using ab65302 kit (Abcam) according to the manufacturer’s protocol. Each fraction was incubated with the cathepsin-D substrate GKPILFFRLK(Dnp)-D-R-NH₂ labeled with MCA at 37 °C for 1 h in the dark. Fluorescence was measured at Ex: 328 nm, Em: 460 nm, in a solid white 96-well plate (Costar). Citrate synthase activity was assayed using ab239712 kit (Abcam) according to the manufacturer’s protocol, and absorbance at 412 nm was measured in clear bottom 96-well plate (Costar). Catalase activity was assayed using ab83464 kit (Abcam) according to the manufacturer’s protocol, and absorbance at 570 nm was measured in clear bottom 96-well plate (Costar). Synergy H1 Hybrid Reader was used for all three enzyme assays.

To assess the secretion efficiency of the transformants, 96 colonies from the selection plates were evaluated as described in S7 Fig. We picked transformed colonies and cultured in 500 μL of TAP medium for 7 d in deep-well plates (Corning Axygen®, No.: PDW500CS, Thermo Fisher Scientific Inc., Waltham, MA), covered with Breathe-Easy® (Sigma-Aldrich®). Cultivation was performed on a rotary shaker, set to 150 rpm, under constant illumination (50 μmol photons/m²s). Then, 100 μL of each sample was transferred to a clear bottom 96-well plate (Corning Costar, Tewksbury, MA, USA) and fluorescence was measured using an Infinite® M200 PRO plate reader (Tecan, Männedorf, Switzerland). Fluorescence was measured at excitation 575/9 nm and emission 608/20 nm. Supernatant samples were obtained by spinning deep-well plates at 3000 × g for 10 min and transferring 100 μL from each well to the clear bottom 96-well plate (Corning Costar, Tewksbury, MA, USA), followed by fluorescence measurement. No normalization was conducted for any mCherry fluorescence, but the chlorophyll contents were checked to infer successful cell growth. 10.17504/protocols.io.kfnctme

For the inhibition of HCMV entry study, a neutralization assay was performed as previously described [21 (link), 25 (link), 28 (link)]. Briefly, ARPE-19 cells were seeded at 1.5x10⁴ cells/well in a clear-bottom 96-well plate (Corning). Approximately 24 h later, the medium in every plate was replaced with 50 μl per well of fresh growth medium. Purified PC, NAb 62–11 and 1B2 [21 (link)] were 2-fold serially diluted starting from 300 μg/ml, 100 μg/ml and 2 μg/ml respectively. PC and NAb dilutions were mixed with complete growth medium containing approximately 9,000 PFU of HCMV TB40/E and incubated for 2h at 37°C. The mixture was transferred to the cells in duplicate wells. After 48 hours, cells were fixed and IE-1 immunostaining performed using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. NAb concentration inhibiting 50% of the virus infectivity (IC50) was also calculated as previously described [21 (link)]. For the analysis of the HCMV-specific NAb response in immunized mice, the same protocol was used. Mouse serum starting dilution was 1:50. For the evaluation of HCMV NAb in human serum products, depleted and mock-depleted IvIg starting dilution was 50 μg/ml, while depleted and mock-depleted human serum dilution started from 1:200.