Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Illustration of the geometry of the nanostraw device and of intracellular delivery using the chip. ( a ) Three nanostraws (orange) interface a cell on top. Cargo (red) can enter the cell through the central nanostraw, which penetrates the cell membrane. ( b ) Three-dimensional sketch of the geometry of a nanostraw device. The nanostraws are anchored in a thin plate (green) which is suspended above a fluidic channel to which liquids and cargoes can be applied from the backside. The drawings are simplified and not to scale. ( c ) Sketch of the locations of nanostraw arrays on the device surface. Based on Ref

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques: Membrane

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: SEM images of PMA-treated THP-1 cells on top of nanostraw chips after cutting through both with a FIB. ( a ) Cross-section of an array of 20 * 20 nanostraws covered by an individual cell. ( b ) Cell on top of a less dense nanostraw array compared to the one shown in ( a ). ( c ) Close-up on one of the nanostraws from ( b ) which is filled with cell contents. ( d ) Interface between an individual cell and a single nanostraw. Based on Ref

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques:

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Analysis of cell viability and function on nanostraw array. ( a ) Bright field and ( b ) immunofluorescence images of THP-1 cells during and after cultivation on nanostraw chips. ( a ) THP-1 cells have been cultivated for 48 h on nanostraws before they were detached. Afterwards, they were cultured standard plastic cell culture plates in the presence of 21 nM PMA and reattached to the bottom of the plates, showing a normal, cell type specific morphology. Scale bars represent 100 μm. ( b ) THP-1 cells have been cultivated with 21 nM PMA for 48 h on nanostraw chips. Staining with 10 µM calcein redorange revealed an excellent overall viability. Scale bar represents 500 μm. ( c ) Immunofluorescence images of THP-1 cells cultivated on nanostraw chips that were incubated with hypoxia green before cells were seeded onto the surface. Cells were further incubated with PMA overnight and cultured for 4 h under physoxic (8% O 2 ) and severely hypoxic (0.1% O 2 ) conditions. Cells incubated under hypoxic conditions (images in the right) are stained by hypoxia green. Upper images show the complete nanostraw array (scale bar: 1 mm), the white boxes indicate the areas that are shown in more detail in the lower images (scale bar: 200 μm). Representative images for four independently analysed arrays

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques: Immunofluorescence, Cell Culture, Staining, Incubation

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Immune cells are not activated by culture on nanostraws. Monocytic THP-1 cells have been stimulated with 21 nM PMA and seeded on either plasticware (6 well plates) or nanostraws for 24 h. ( A ) Neither proteins of the MEK/ERK pathway (phosphorylated MEK1/2) nor the NF-κB pathway (phospho-p65) were induced by cell culture on nanostraws. ( B ) Supernatants of the cells cultured on either 6 well plates or nanostraws were subjected to ELISA analysis. Culture on nanostraws did not increase the secretion of inflammatory cytokines IL-1β, IL-6 or IL-12(p70) compared to plastic control. LPS as a positive control induced a robust increase in IL6 release (data from 2 timely independent experiments (each with two independent biological replicates and a total of 4 different nanostraw arrays resulting in n = 4 each for nanostraws/plastic and for LPS) are given as means ± SD of n-fold induction of cytokine release normalized to plasticware control; data were analyzed by one-way ANOVA plus Tukey-test, ****: p < 0.0001.). Interestingly, the IL-1β release was stimulated in all samples (≈ 500 pg/µL medium), which can be explained by the preceding PMA treatment of the cells . The absolute values of IL-6 and IL-12(p70) were almost undetectable in the controls (≈ 1pg/µL for IL-6 and ≈ 7 pg/mL for IL-12)

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Positive Control

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Substantial evidence of succesful contact of nanostraws with the cytosol of THP-1 cells. SEM and FIB/SEM images of THP-1 cells on nanostraws with areas allowing fluidic contact to a sub-array reservoir containing 0.1 nMol of CTAB stabilized, positively charged Au-nanoparticles (Au-NPs) with 50 nm diameter. Arrays have been incubated top-down for 15 min to optimize delivery through the nanostraws and limit particle uptake via phagocytosis. ( a ) SEM-generated top view of fixed, resin-layered THP-1 cells on the nanostraw array surface, with ( b ) as a zoom-in of the marked region in (a). ( c ) FIB-SEM generated image illustrating the fluidic contact of the three nanostraws at the left (area 1) with more details presented in the zoom-in ( d ). Interestingly, the gray scale of the material seen within these nanostraws is highly comparable to that of the cytosol, strongly pointing towards a successful intracellular contact of the three left nanostraws. In addition to that, Au-NPs exhibit a high contrast in FIB-SEM images appearing as bright structures in the following images. Arrows mark signals that are most likely caused by Au-NPs delivered through the nanostraws. Au-NPs seem to accumulate at the backside of the nanostraw array as a string of pearls (arrow below number 2), which might be favoured by the top-down incubation. ( e ) illustrates a different cell on an independently incubated array (with again more details shown in ( f )). Arrow number 1 marks bright structures in an enclosed intracellular structure, which correspond most likely to lipid drops. The more spherical, bright structure marked by arrow 2 in turn might be caused by nanostraw-delivered Au-NPs. ( g ) and ( h ) are exemplary images for Pb-sucrose treated THP-1 cells incubated on nanostraw arrays with fluidic contact to an underlying reservoir containing a fluorescence-labelled antibody towards intracellular α-tubulin (red signal). Counterstaining with Calcein AM (green) marked all viable cells. The white encircled areas are linked to the fluid reservoir below the arrays. In these areas, we observed double-positive cells, which are likely to have administered the red fluorescence via nanostraws delivery

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques: Incubation, Generated, Fluorescence

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Functionalisation of the nanaostraw array: APTES coating of the array surface to immobilize protein on the nanostraws. Immunofluorescence images of different nanostraw arrays: either uncoated (above) or coated with APTES with subsequent incubation with biotin and FITC-labelled streptavidin. The arrays coated with APTES showed a much higher fluorescence due to immobilized FITC-streptavidin than uncoated arrays. The immobilization is much more pronounced on the nanostraws themselves compared to the surrounding SiO 2 surface. Representative images of four independently analyzed nanostraw arrays, scale bar: 1 mm

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques: Immunofluorescence, Incubation, Fluorescence

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Protein and mRNA expression of typical ion channels in THP-1 cells. Monocytic THP-1 cells have been stimulated with PMA overnight (under exactly the same conditions as for the nanostraw experiments). After that, cells were incubated under normoxia (24 h; NOX) or under hypoxic conditions (HOX: 1% O 2 ) for 2 h, 4 h, and 24 h respectively. ( a ) mRNA expression of TRPM2 , TRPM4 and KCNA3 (encoding Kv1.3) under the given conditions. Whereas TRPM2 showed a significant downregulation after 24 h of hypoxic incubation, TRPM4 mRNA was upregulated. KCNA3 mRNA showed no changes (mean ± SD; n = 9; one way ANOVA plus Dunnett´s multiple comparisons test; ***: p < 0.001, ****: p < 0.0001). ( b ) TRPM4 and Kv1.3 protein are expressed in THP-1 cells under normoxic conditions and can be found in the cytosol and membrane of the cells (400x magnification, scale bar: 20 μm) but did not show altered expression under hypoxia (data not shown). ( c ) TRPM2 protein was cleaved under hypoxic conditions and shorter versions of the protein became detectable after only 2 h of hypoxia. 50 µg of whole cell lysate have been applied to Western blotting. Blot is representative for three independent results. ( d ) Intracellular protein distribution analysis of TRPM2 showed a prominent overall downregulation of TRPM2 after 2 h of hypoxia especially in areas close to the cell membrane. This effect seemed to recover over time (400x magnification, scale bar: 20 μm). Negative control (without primary antibody) did not show staining for TRPM2 at all

Article Snippet: Chips with nanostraw arrays were activated with 2.5% glutaraldehyde (#sc-257558, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h and then incubated with 10 μM Hypoxia Green (# I14834 , Life Technologies, Darmstadt, Germany) in aqueous phosphate buffer for 1 h. After extensive washing with PBS, THP-1 cells were seeded (with 21 nM PMA) onto the functionalized chips and cultivated the other day under 8% and 0.1% ambient oxygen for 4 h. Cells were fixed with 4% paraformaldehyde solution under the respective oxygen conditions and analyzed with the Zeiss Axio Imager.M2 fluorescence microscope (Zeiss, Oberkochen, Germany).

Techniques: Expressing, Incubation, Membrane, Western Blot, Negative Control, Staining