Glass microfiber filter

Exponentially growing cells raised in minimal MOPS medium (Wang et al., 2007b (link)) supplemented with either 1% arabinose or glucose, at 30°C, were split at OD 0.2 into equal 1.2 ml cultures. Cells continued to grow until they reached OD 0.3., at which point 30 µg/ml rifampicin was added to one of the cultures for 2 min. Next, 38 µCi ³H-thymidine (Perkin Elmer 70–90 Ci/mMol) was added to both cultures, and timepoints were taken at 2 min intervals by pipetting 200 µl of cells into 3 ml of ice-cold 10% TCA. Samples were collected on glass microfiber filters (GE Healtthcare #1825–025), and washed 3x with 5% TCA prior to detection on a liquid scintillation counter.

A 1.0 mL aliquot was taken from each strain (n = 3) after 45 days of growth and 72 h +/− UVA+UVB light exposure. These aliquots were filtered through a glass microfiber filter (47 mm, GE Life Sciences) under vacuum. Each filter was stored in amber Eppendorf tubes and frozen at −80 °C until extraction, which was performed as described by Strickland and Parson [42 ]. Total carotenoid content was calculated by the method of Kirk and Allen [43 (link)].

The dispersions were characterized
by DLS (Zetasizer Nano Series, Malvern Instruments) to obtain the
particle diameter. Dispersions were diluted with deionized water and
filtered (GE Healthcare, glass microfiber filter with polypropylene
housing, pore size of 2 μm) before measuring. The dry solid
content (ASR included) was determined gravimetrically by the difference
in the weight of the wet and dry dispersions. Approximately 1 g of
the dispersion (wet) was applied on a filter paper, the dispersion
dried in an oven at 140 °C for 20 min, and the solid residue
weighed.

Trp uptake experiments were performed as previously reported [19 (link),38 (link)]. In detail, the HeLa cells were extensively washed (3×) in PBS (pH 7.4), and then resuspended in uptake assay buffer (PBS plus 0.3 mM MgCl₂) to give 1 × 10⁶ cells/mL. [³H]Trp (150 nM) was added to a 0.3 mL aliquot of cell suspension plus or minus 500 nM TrpRS protein. Cellular uptake of Trp was then monitored at 25 °C over a series of timepoints (0, 1, 2, 3, and 4 min). Specifically, 50 μL samples were subjected to rapid vacuum filtration through a glass microfiber filter (GE Healthcare Life Sciences) with a particle retention of 1.2 μm. Filters were washed (5×) with 5 mL PBS, air-dried, and analyzed on a scintillation counter (PerkinElmer, Waltham, MA, USA). Trp uptake increased over time in a linear fashion. Uptake was found to be linear, and the initial rate was calculated (fmol min⁻¹).

Blood was collected with heparin as an anticoagulant and centrifuged at 2,400 g for 5 min at room temperature, followed by aspiration of plasma and buffy coat. Packed erythrocytes were purified using percoll gradients to remove reticulocytes. The mature erythrocytes were washed three times with PBS and re-suspended to 4% hematocrit. The uptake assay started with transferring 54 µl of erythrocyte suspension to an Eppendorf tube with 6 µl C₁₄-glucose (PerkinElmer, Waltham, MA) master mix (50 mM adenosine with 1 mCi∙ml⁻¹ C₁₄-glucose in PBS) to get a final glucose concentration of 5 mM. The uptake was performed for up to 120 min and stopped by adding 100 ml cold stop solution (0.9% saline), then centrifuged at 2,400 g for 5 min. The supernatant was withdrawn and the erythrocyte pellet was lysed in 60 µl water and the lysate was spread on a glass microfiber filter (GE Healthcare Life Sciences, catalogue number: 1825-025), heat-dried for counting of C₁₄ isotope using a scintillation counter (LKB WALLAC 1209 EACKBETA Liquid Scintillation Counter, LKB Instruments, Victoria, Australia). Also, 54 µl of erythrocyte suspension (washed and re-suspended to 4% hematocrit as mentioned above) was aliquoted for total protein measurement using Pierce BCA Protein Assay kit (Thermo Scientific, catalogue #: 23225, Rockford, IL, USA).