rhod pe  (Croda International Plc)

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: a: Two populations of synthetic cells are mixed. Population 1 contains T7 RNA polymerase and TxTl machinery expressing αHL. Population 2 contains the plasmid encoding GFP under the control of a T7 promoter and Ni-NTA lipids in the membrane. The His tag on the αHL (in population 1) binds to the Ni-NTA lipids (in population 2), inducing membrane fusion. After lumens mixed, the T7 RNA polymerase from population 1 enables transcription and expression of GFP from a plasmid in population 2. Different ratio of αHL plasmid and NTA tag: b and c : The fusion efficiency depends on both the amount of the αHL plasmid in population 1, and the concentration of the Ni-NTA lipids in population 2. Fusion efficiency is measured as membrane mixing represented by FRET intensity between Cy5 and Cy7 ( b , more Cy5 fluorescence correlates with more fusion), and lumen mixing represented by GFP expression ( c , more GFP fluorescence correlates with more fusion). Individual data points and statistical analysis for GFP fluorescence is on , for Cy5 FRET fluorescence on . After fusion, selected samples were purified on a size exclusion column to measure content leakage, data on . A calibration curve for all FRET membrane mixing measurements is on . Data for the stability of the liposomes with membranes containing a different percent of Ni-NTA lipids are on and the data for the stability of liposomes with an increasing amount of αHL are on . Different ratio of cells: d - h : Example of individual time courses of GFP expression in fusion experiments. d - f : The amount of either population 1 containing αHL or population 2 containing GFP plasmid was varied. 1x is 1mM total lipid concentration and 2x is 2mM total lipid concentration. g : “No fusion” conditions indicate a lack of Ni-NTA lipids on population 2. h : The positive control uses DNA fusion tags instead of the αHL and Ni-NTA pair. Shaded area indicates SEM, n=3 independently prepared and processed liposome samples. The Y axis on panels in this section (except negative control g ) is the same, to allow for quick visual comparison of the data. i : Fusion induced GFP expression depends on the ratio of population 1 containing αHL vs. population 2 containing GFP. All values are averaged with n=3 independently prepared and processed liposome samples. Individual GFP fluorescence data points and statistical analysis are on . j and k : RT qPCR and qPCR measurements of the GFP gene, l : GFP fluorescence from the same fusion samples as j and k with all measurements taken after a 12-hour incubation. The graphical legend below panels j , k , and l marks the ratio of population 1 (αHL, T7 RNAP) to population 2 (GFP plasmid, Ni-NTA) and the presence or absence of a fusion event (absence of fusion means the absence of Ni-NTA lipids in population 2). Box and whiskers data points on panels j and k are prepared with n=3 independently prepared and processed liposome samples. Individual data points and statistical analysis are on . Dots on top of bars on panel L indicate all individual data points, the bars are the average value. m : Two examples of microscopy images of liposomes after fusion. The green channel shows GFP expression, the red channel shows Rhodamine membrane dye, and the third panel in each grouping is composite of the red and green channel. The liposomes are from experiments under the same fusion conditions as data on panel d , with images collected after 12h of incubation. All protein expression data on this figure were collected using whole cell lysate (TxTl).

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Expressing, Plasmid Preparation, Control, Membrane, Concentration Assay, Fluorescence, Purification, Liposomes, Positive Control, Negative Control, Comparison, Quantitative RT-PCR, Incubation, Microscopy

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: a - h : Fusion efficiency controlled by strength of αHL expression . Fusion between two populations of synthetic cells, according to the same setup as shown on , with lumen fusion indicated by GFP expression and membrane fusion indicated by Cy5 signal. a - c : Flow cytometry analysis of samples prepared with either T7 αHL (panel a ) or T7Max αHL (panel b ), after fusion incubation. Full flow cytometry data are on . c : summary of the flow cytometry results, showing percentage of liposomes expressing GFP in samples with “weaker” T7 αHL and with “stronger” T7Max αHL. All plasmids other than αHL were under the T7 promoter, the T7Max was only used in αHL plasmid. “T7Max cells” indicate cells with αHL under T7Max, with all other plasmids in that cell under regular T7. d - h : characterization of liposome fusion with T7 αHL, T7Max αHL, and negative control without αHL. d : GFP signal (green bars) indicating lumen mixing, and % membrane mixing (gray bars), measured after 12h incubation. e and g : abundance of plasmid DNA ( E for αHL plasmid and G for GFP plasmid) in the same fusion experiments as shown on panel d . f and h : abundance of mRNA ( F for αHL gene and H for GFP gene) in the same fusion experiments as shown on panel d . i - o : Selection over 5 generations of cell cycle. All samples were prepared as described in the cell cycle experiments shown on Figure 3 . I - m : Flow cytometry analysis after 5 generations of cell cycle with two competing populations in each sample. Four types of samples were prepared: cells with T7 αHL plasmid (symbol ), cells with T7 αHL plasmid and GFP plasmid (symbol ), cells with T7Max αHL plasmid (symbol ), and cells with T7Max αHL plasmid and GFP plasmid (symbol ). Two types of cells were mixed, at equal ratio, for each experiment. In each case, T7 and T7Max cells were mixed, with one of the populations containing GFP reporter. After either 5 generations of complete cell cycle (symbol ), or incubation without DNA replication and without feeding and division (symbol ) samples were analyzed using flow cytometry. Experiments with GFP reporter in T7 αHL cells: after DNA replication and division on panel i , and without DNA replication and division on panel j . Experiments with GFP reporter in T7Max αHL cells: after DNA replication and division on panel l , and without DNA replication and division on panel m . Full flow cytometry data are on . k : summary of the flow cytometry data, showing percentage of liposomes containing GFP in each of the 4 selection samples from panels i , j , l and m . n : Relative abundance of T7 and T7Max promoter sequences in samples after 5 rounds of complete cell cycle. Two types of synthetic cells were prepared for this experiment: cells with T7 αHL plasmid and cells with T7Max αHL plasmid. For each experiment, those two initial populations were mixed at ratio indicated in panel legend (equal 1 to 1 ratios, or samples where T7 population was 90% of the total abundance with the T7Max being 10%). All resulting samples had identical total lipid concentration, but different ratios of T7 to T7Max populations. After either 5 generations of full cell cycle (symbol ), or incubation without DNA replication and without feeding and division (symbol ) the abundance of T7 and T7Max sequences was quantified via Illumina sequencing (Methods section 16, and and ). o : selection experiments with all cells carrying a fluorescent protein of similar size (normalized metabolic load). Experiments performed similarly to those on panels i - m , but this time both T7 and T7Max cells carried fluorescent protein (GFP or mCherry, as indicated in the legend under graph on panel o ). After 5 generations, fluorescence of both GFP and mCherry was measured, and normalized to calibration curves indicating what percent of each population contains this fluorescent protein (see for raw fluorescence data and for calibration curves, for statistical analysis). The red bracket on panels k and o indicates the difference between the percent of population containing T7 and T7Max (all from starting 1 to 1 ratio). Experiments under similar set of conditions are indicated with yellow symbol: samples with GFP in T7 αHL population, and samples with GFP in T7Max αHL population. Data on all panels represent n=3 of independently prepared and processed liposome samples. Dots on top of bars indicate individual data points, the bars are the average value. Statistical analysis of data presented on this figure is in . All protein expression data on this figure were collected using PURE system.

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Expressing, Membrane, Flow Cytometry, Incubation, Liposomes, Plasmid Preparation, Negative Control, Selection, Concentration Assay, Illumina Sequencing, Fluorescence

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: Liposome fusion experiment monitored by FRET effect, measuring fluorescence of donor dye from the FRET pair. In synthetic cell fusion experiments, one population of cells contains both FRET donor and FRET acceptor, while the other population does not contain any FRET dyes. Before fusion, the FRET dyes are in relatively close proximity, resulting in high FRET effect, which means low donor fluorescence signa and higher acceptor fluorescence signal. After fusion and membrane mixing, the FRET dyes are “diluted” in the now larger membrane, putting the FRET dyes further apart from each other on the larger membrane surface. The efficiency of the FRET effect decreases, which results in increase of donor fluorescence and decrease of acceptor fluorescence. In the fusion experiments in this work, two different pairs of FRET dyes were used. Donor NBD - acceptor Rhodamine, this dye pair was used when no concurrent GFP monitoring was needed (since NBD fluorescence spectrally overlaps with GFP) Donor Cy5 – acceptor Cy7, this dye pair was used to allow for monitoring of GFP fluorescence at the same time. In all FRET experiments, we monitored the fluorescence of the donor dye. Increased donor fluorescence indicated increased membrane mixing.

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Fluorescence, Membrane

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: Synthetic cells were created with GFP plasmid. The cells also contained 1mM of Cy5.5. After 12h incubation at 30°C, cells were purified (methods section 27) to measure leakage of the small molecule dye. Each panel shows one purification trace, label indicated type of cells (GFP containing cells on this figure), type of readout of fluorescence (GFP channel or Cy5.5 channel, see for list of wavelengths used), and replicate sample number (3 independent samples). This data was collected using PURE pREP.

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Plasmid Preparation, Incubation, Purification, Fluorescence

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: Individual Cy5 FRET fluorescence data points for the heatmap on . Error bars indicate SEM, n=3 independently prepared and processed liposome samples. Statistical analysis shows P values for each series of NTA concentrations compared to 0mM αHL plasmid. ns = P > 0.0; * = P ≤ 0.0, ** = P ≤ 0.01, *** = P ≤ 0.001. This data was collected using TxTl.

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Fluorescence, Plasmid Preparation

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: A : Two populations of synthetic cells were mixed, population 1 containing VAMP transmembrane domain labeled with His tag and encapsulating T7 RNA polymerase, and population 2 containing Ni-NTA lipid tags and encapsulating plasmid with GFP under T7 promoter. Only if the lumen of the membranes fuses, GFP could be expressed (T7 RNA polymerase from population 1 is needed to express GFP plasmid in population 2). B : Lumen fusion, measured as expression of GFP, for experiments with varying amount of VAMP protein and varying amount of NTA lipid (described as mol% of total lipid). Higher GFP fluorescence means more lumen mixing. C : Membrane mixing, measured as FRET interaction between Cy5 and Cy7 FRET pair (see for general scheme of FRET experiment). Higher Cy5 fluorescence means more membrane mixing. Population 2 was labeled with 0.1mol% of each Cy5 and Cy7 lipid anchored dyes. Individual heat map data points for GFP are on , and individual heat map data points for Cy5 are on . This data was collected using TxTl.

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Labeling, Plasmid Preparation, Expressing, Fluorescence, Membrane

Journal: bioRxiv

Article Title: A Chemically Defined Synthetic Cell Capable Of Growth And Replication

doi: 10.64898/2026.07.01.735724

Figure Lengend Snippet: Fusion between two populations of synthetic cells, one carrying T7RNAP and the other population carrying GFP DNA and Ni-NTA lipid tag. Lumen fusion was monitored by GFP expression (panel A) and membrane mixing was monitored by Cy5 FRET signal (panel B). Individual heat map data points for GFP are on and individual heat map data points for Cy5 are on . After fusion, selected samples were purified on size exclusion column to measure content leakage, data on . This data was collected using TxTl.

Article Snippet: The dyes used to label liposomes in this project, and the stock amounts used to prepare sample of 30μl of 1mM liposomes, were: NBD (NBD-PE, (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher N360), 2.86μl stock for each thin film; Rhodamine (LissamineTM Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Thermo Fisher L1392), 4.0μl stock for each thin film; Cy5 (Cy5-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5), Avanti 810335C), 3.79μl stock for each thin film; Cy7 (Cy7-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7), Avanti 810337C, 3.98μl stock for each thin film.

Techniques: Expressing, Membrane, Purification

Journal: Molecular Neurodegeneration

Article Title: Obesity-driven phosphatidylethanolamine dysregulation impairs neuroimmune crosstalk and accelerates Alzheimer’s pathogenesis

doi: 10.1186/s13024-026-00943-3

Figure Lengend Snippet: The PE high flux exacerbates the pathologies of AD. A . schematic representation of the generation of engineered EVs, designed to be free of endogenous nucleic acids and proteins, and derived from EVs isolated from lean and obese adipocytes. B . Quantification and size analysis of engineered EVs derived from adipocytes, performed by nanoparticle tracking analysis using a NanoSight system with a 1:1000 dilution. C . Representative biodistribution of engineered EVs incorporating Cy5 PE after intraperitoneal (I.P.) injection in WT mice. The images show the following conditions: (1) brain with PBS administration, serving as the control; (2) brain with Cy5 18:1 PE administration; (3) brain with engineered EVs lacking Transferrin for blood-brain barrier (BBB) crossing; and (4) brain with engineered EVs modified with Transferrin to facilitate BBB crossing. D . ELISA assay of PE levels of the nature EVs from lean and obese adipocytes. E . ELISA assay of PE levels of WT mouse brains with/without the indicated EV treatment. F . ELISA assay of PE levels of 5XFAD mouse brains with/without the indicated EV treatment. G . Schematic representation of the generation of engineered EVs, derived from lean adipocyte–derived EVs and supplemented with additional PEs, designed to be free of endogenous nucleic acids and proteins. H - I . Comparison of the mean levels of the indicated AD pathological proteins in the brains of 5XFAD and age-matched control mice following long-term administration of vehicle, PE low -EVs, and PE high -EVs, measured by ELISA. ( D ), data are derived from four independent experiments, each treated as a biological replicate for statistical analysis. ( E , F , H and I ), each data point represents an individual mouse, and individual mice were treated as biological replicates for statistical analysis. Each group consisted of 6–14 mice, with approximately equal numbers of males and females. Data are presented as mean ± SEM; *, increase; #, decrease; */ # p < 0.05, **/ ## p < 0.01, and ***/ ### p < 0.001. Statistical significance was determined using paired one-tailed t-tests for (D and F, right) and unpaired two-tailed t-tests for ( E , F left, H , and I ), with obese-EV and lean-EV groups each compared separately to the PBS control and no direct comparisons made between obese-EV and lean-EV groups

Article Snippet: To generate PE-enriched EVs, additional PE, including 16:0–18:1 PE (Avanti, 850757P), 16:0–20:4 PE (Avanti, 850759C), C18 (Plasm)-18:1 PE (Avanti, 852758P), C18 (Plasm)-20:4 PE (Cayman, 37,137), and/or 18:1 Cy5 PE (Avanti, 810335C, for imaging purposes), was incorporated into the organic phase.

Techniques: Derivative Assay, Isolation, Injection, Control, Modification, Enzyme-linked Immunosorbent Assay, Comparison, One-tailed Test, Two Tailed Test