Sodium dithionite

Sodium chloride (NaCl), dipotassium hydrogen phosphate, potassium dihydrogen phosphate (KH₂PO₄), imidazole, potassium ferricyanide, sodium dithionite, phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), d(+)-glucose, d-serine, sodium l-lactate, sodium hydroxide (NaOH), sulfuric acid (H₂SO₄), 25% glutaraldehyde, and 1% (w/v) Nafion^TM were purchased from MilliporeSigma (Burlington, MA, USA). Mesoporous carbon particles (Cnovel P(4)050) were purchased from Toyo Tanso (Tokyo, Japan). All other chemicals were of reagent grade. Platinum wires were purchased from TANAKA KIKINZOKU (Tokyo, Japan). Gold disk (7 mm²) and silver/silver chloride (Ag/AgCl) reference electrodes were purchased from BAS Inc. (Tokyo, Japan).

N-(2-Hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) (HEPES) was purchased
from Fisher Scientific. Imidazole was purchased from J. T. Baker Chemical
Co. Potassium chloride and glycerol were purchased from EMD Chemicals.
β-Mercaptoethanol (BME), sodium dithionite, sodium sulfide,
5′-deoxyadenosine (5′-dAH), and S-adenosylhomocysteine
(SAH) were purchased from MilliporeSigma. Kanamycin, ampicillin, dithiothreitol
(DTT), arabinose, isopropyl β-d-1-thiogalactopyranoside
(IPTG) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were
purchased from Gold Biotechnology. Ni-nitrilotriacetic acid (NTA)
resin was acquired from Qiagen. SAM was synthesized and purified as
described previously.^{45 (link)} DNA isolation kits
were purchased from Macherey-Nagel (Dueren, Germany). All other chemicals
and materials were of the highest grade available and were from MilliporeSigma.

Flippase was measured as previously described [14 (link)]. Briefly, cells were resuspended in 3ml of flippase assay buffer and NBD-PS (Avanti Polar Lipids) was added to a final concentration of 3 μM. Cells with NBD-PS were divided into aliquots and incubated for 0, 1, 5, 15, 30 or 45 minutes. After each incubation time, half of the cell suspension was separated for non-extracted sample and kept on ice. The remaining half was spun down to remove non inserted NBD-PS and subjected to BSA extraction of NBD-PS from the outer leaflet by adding 3% fatty acid free BSA (MP Biomedicals) in flippase assay buffer. After incubation on ice for 20 minutes, freshly prepared sodium dithionite (Sigma) was added to a final concentration of 10 mM and incubated a further 10 minutes, to reduce the NBD lipids in the outer cell surface. The cells were spun down and resuspended in flippase assay buffer containing 0.25% BSA and 2 μg/ml PI. NBD-PS signal from unextracted and extracted samples was measured by flow cytometry from living cells after exclusion of PI-positive dead cells, using a BD Fortessa. Non-extractable NBD-PS in the BSA and sodium dithionite treated sample was presented as the percentage of total amount in the control unextracted sample. Scramblase was measured in the same way but with NBD-PC (Avanti Polar Lipids).

Capsaicin and A967079 were purchased from HelloBio (Bristol, UK), BCTC was purchased from Tocris (Wiesbaden-Nordenstadt, Germany). Ruthenium red, α1-antitrypsin, protoporphyrin-IX, hemin, iron(II) sulfide, sodium dithionite, DL-dithiothreitol, chelerythrine, reduced glutathione, and carvacrol were purchased from Sigma-Aldrich (Taufkirchen, Germany). hemin was resuspended in 30 mM KOH to produce a 10 mM stock solution which was stored on ice. The dilutions needed for the measurements were freshly prepared before use and dissolved in external solution.