Bartosh et al. · Cells · 2025

New Approaches to Old Techniques in Cell Handling for Microscopy

Zhanna Bartosh, Veenu Aishwarya, Wayne W. Hancock, Tatiana Akimova

The study

What was asked, and what was found

Bartosh et al., Cells, 2025, is a methods paper, and AUM BioTech co-authored it. The question was practical rather than biological: how do you get primary immune cells and adherent cell lines onto a microscope slide, in a long time course, without a cytocentrifuge and without the cost of commercial chamber slides. The answers are a smear and drying protocol on charged slides, and a chamber slide made by punching holes in parafilm and melting the strip onto the glass, which gave wells of roughly 30 microlitres from a 6 millimetre punch.

The worked example is uptake of a far red labelled AUMsilence scramble control oligo at 1.5 μM, followed by microscopy at 15, 30, 60, 90, 120, 150 and 180 minutes. In mouse splenocytes, roughly 80% of cells showed oligo inside them by 15 minutes, and the commonest picture across the whole time course was a diffuse low intensity signal rather than bright puncta. The fraction with no detectable uptake fell to 3.5% at 120 minutes. The two adherent lines behaved differently again: A549 and HEp-2 both reached 100% of cells by 15 minutes and held the signal for at least 3 hours, but A549 put small granules at the cell periphery early and gave a bright nuclear signal, while HEp-2 stayed diffuse and built large cytoplasmic granules instead.

One condition governs how these numbers may be read, and it is in the Methods rather than the Results. The uptake medium was supplemented with 3 mM ammonium chloride and 1 μM arsenic, which the authors state was done to enhance gymnosis. Every percentage above was measured in that medium. The study is therefore strong evidence that this microscopy workflow resolves where an oligo goes inside single cells over hours, and it is not evidence about uptake in ordinary culture medium. It is also an uptake study rather than a knockdown study: nothing was silenced, and no gene was targeted.

Key findings

In the study's uptake medium, about 80% of mouse splenocytes carried detectable oligo inside them after only 15 minutes.(Results, 3.2, Fig. 6)
Both adherent cell lines took up the oligo in every cell by 15 minutes and held it for at least 3 hours.(Results, 3.3)
The signal was diffuse rather than punctate at early times, which the authors read as the oligo not being trapped in lysosomes and reaching the nucleus of primary cells evenly.(Results, 3.2)
The two cell lines handled the same oligo differently over 3 hours, which is the paper's argument for validating any new compound in more than one line.(Results, 3.3)
Among the splenocytes, cells with neutrophil morphology behaved least like the rest, ending with the oligo concentrated in large granules and kept out of the nucleus.(Results, 3.2, Fig. 5A)
The method the paper is about is a cheap chamber slide made from parafilm and a hole puncher, and a slide preparation route that removes the need for the centrifuge, which is what made a seven point time course of oligo uptake practical. The paper still allows a brief gentle spin, 3 minutes at 300 g, when a sample has to be concentrated.(Methods, cell smear preparation)

Product: AUMsilence sdASO
Models: mouse splenocytes, including monocytes and macrophages, lymphocytes and neutrophils · primary cellsA549 human lung carcinoma and HEp-2 · cell lineHEp-2, identity noted by the authors as a HeLa derivative · cell line
Delivery: added to the cells in medium with no transfection reagent, but in an uptake medium the authors supplemented with ammonium chloride and arsenic to enhance gymnosis
Readouts: fluorescence microscopy, immunofluorescence staining for beta-actin and histone H3, propidium iodide and DAPI nuclear staining, flow cytometry grade surface marker staining, two-sided Fisher's exact tests with Bonferroni correction
Measured: oligo concentration used in every uptake experiment: 1.5 μM (in the supplemented uptake medium, for both suspension and adherent cells)mouse splenocytes showing intracellular oligo signal: approximately 80% (15 min, 1.5 μM, in the supplemented uptake medium)mouse splenocytes with no detectable uptake, over time: highest at 15 min, minimum 3.5% at 120 min, 13% at 180 min (1.5 μM, in the supplemented uptake medium)mouse splenocytes carrying large intracellular granules of oligo: 8% (at 120 min and later, alongside a fall in the intensity of the diffuse signal)uptake in the two adherent cell lines: 100% (as early as 15 min, sustained for at least 180 min, 1.5 μM, in the supplemented uptake medium)A549 cells showing nuclear exclusion of the oligo: 5.6% at 60 min, 3% at 150 min, undetectable at 180 min (time course at 1.5 μM)HEp-2 cells showing partial nuclear exclusion of the oligo: 1.5% at 60 min, 4% between 90 and 150 min, 1.8% at 180 min (time course at 1.5 μM)HEp-2 cells showing bright nuclear signal: 14% at 90 min rising to 39% at 150 min (time course at 1.5 μM)working volume of the home made chamber well: approximately 30 μL from a 6 mm hole (parafilm and single hole puncher chamber slide)
Figures: Figure 5 Mouse splenocyte types identified by morphology, monocytes and macrophages, lymphocytes and neutrophils, and the range of oligo distribution patterns seen in primary cells.Figure 6 The splenocyte time course: what lymphocytes look like at each time point, and the percentage of cells in each distribution pattern from 15 to 180 minutes.Figure 8 The A549 time course, including the peripheral granules at 15 minutes, the bright nuclear signal through the first 2 hours, and the small fraction of cells excluding the oligo from the nucleus.Figure 9 The HEp-2 time course, the comparison of large cytoplasmic granules against A549, and the perinuclear accumulation seen by 180 minutes.
Citation: Bartosh et al. New Approaches to Old Techniques in Cell Handling for Microscopy. Cells 2025;14:1271. doi:10.3390/cells14161271

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