Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: The i3C and its main components: (1) - cylindrical sample chamber with a bottom made of glass picowells substrate (B and C - SEM micrographs), (2) - elevated flow conduit and its front basin, (3) - two engraved longitudinal grooves, (4) - the sliding cover slip (orange), (5) - waste accumulation area . Note the reference mark pointed to by the yellow arrow in panel C.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques:

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: Attachment of the glass picowells substrate to the i3C base . (A) The base (1) is placed upside down, and the picowells substrate (2) is gently placed in its socket (3, niche) with its picowells facing down the lower entrance of the cell chamber (4). Next (B): Medical grade UV adhesive (dashed blue) is gently injected in the empty space created between the chip edge and the niche wall, then irradiated and cured. Then, a small drop of liquid PDMS (green) is gently poured near the free edges, and is drawn by capillary forces to fill the empty space between the peripheral face of the chip and the bottom of the niche. The PDMS does not penetrate into the cell chamber area (yellow) due to lack of capillary forces in this open region. When PDMS is cured, the structure is practically sealed to fluids and withstands freezing - thawing cycles.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques: Adhesive, Injection, Irradiation

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: A) Open cryostage, the lid (1) is detached, the i3C (2, cloudy structure) is attached via its adaptor (3, metal frame around the i3C) to the cooling element (4, circular silver body) . (B): The lid is closed. Dry nitrogen gas pipe is in place (5). The sample in the i3C is ready to undergo a freezing - thawing cycle. Centers of the lid's window (through which images are acquired - light coming from the condenser is seen) and the i3C's bottom are overlapping and situated on the microscope optical axis. (C) Cross section of the i3C placed on the cryo-stage silver cooling element (1) ready for experiment. The i3C polycarbonate body (2) hosts the glass picowells chip (3) which is secured to its place by bonding. Bottoms are aligned for a perfect contact with the flat cryo-stage. Cells and media (4) which were previously loaded into the i3C are trapped in the cell chamber and the flow conduit due to the capillary action of the sliding cover slip (5). The cryo-stage lid is then closed so that the center of its window (6) and microscope objective (7) optical axis are overlapping to facilitate microscopic observation of cells in the i3C. Note that for the sake of clarity the colors of the i3C components here match those appearing in Figure 1A.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques: Microscopy

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: Working with the i3C: (A) Packed chip in a sterile pouch . (B) The cover glass is moved back enabling the cell suspension to be loaded into the chamber. The glass is then closed to cover the chamber and to create a short capillary traction area over the input basin (C), so additional aliquots can be added and replace the cell media. Aliquots can be added until the waste area is full and there is no room for additional liquids to enter (D). See text for specific volumes and procedures.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques: Sterility, Suspension

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: Monitoring cell retention during the cell freezing - thawing cycle . U937 cells, labeled with the Hoechst dye (blue fluorescence) and Quantum Dots (red fluorescence), were loaded into the i3C and then underwent a freezing and thawing cycle, which included the following documentation steps: first, before closing the cryostage lid; (A) transmitted light image is superimposed with the fluorescence image of the same field (B). Next, images were taken after closing the cryostage lid (C) and following the pre-cooling step (D), after which freezing was performed. Immediately after thawing, while the lid was still closed, transmitted light (E) and fluorescence (F) images were acquired. To assess the vitality of the same thawed cells, we examined their ability to hydrolyze FDA by exchanging their cryo-media with FDA staining media: 7.5 μm of FDA staining solution was introduced to the conduit basin of the i3C (when the i3C was in the cryostage chamber), and then (after a few minutes) the fluorescence of the FDA-stained cells was imaged (G). Note that the majority of cells retain their spatial location. The red arrow indicates a single cell that changed location after thawing. Bars: 25 μm.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques: Labeling, Fluorescence, Staining

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: Cell retention. Box plot of the percentage of dislodging cells during the freezing - thawing cycle . U937 cells were loaded into the i3C (n = 5), and the same area (20 × 20 picowells) was imaged in the course of cell handling. Total cells dislodging was calculated after each step: 1 - after pre-cooling, 2 - immediately after thawing, 3 - post-thawing after opening the cryostage lid, 4 - after post-thawing medium exchange. The mean value for each data set is indicated by the central line and the first and third quartiles are the edges of the box area.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques:

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: Monitoring cell viability after the freezing (slow cooling rate, 1°C per minute) and thawing cycle . Representative transmitted light (A) and corresponding fluorescent images of FDA (B) and PI (C) staining of U937 cells within the i3C after a freezing and thawing cycle. The red line connects images of the only cell in the field found to be PI-positive. The blue arrow indicates a reference region in the picowell field at the bottom of the i3C chamber. Bars: 25 μm.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques: Staining

Journal: BMC Cell Biology

Article Title: The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

doi: 10.1186/1471-2121-11-54

Figure Lengend Snippet: Cell viability after freezing and thawing within the i3C and in control standard cryo-vials . U937 cell populations underwent slow or fast freezing followed by thawing in either the i3C or cryo-vials, after which they were exposed, while residing in the i3C or the vial, to staining mixture of FDA and PI. The percentages of dead U937 cells (PI positive) are shown. Bars represent mean and SD values for at least 6 experiments. p < 0.05.

Article Snippet: A similar dislodging percentage was found when the cell-loaded i3C was stored in Revco or in a nitrogen storage tank.

Techniques: Control, Staining

Journal: Nature Communications

Article Title: PI(18:1/18:1) is a SCD1-derived lipokine that limits stress signaling

doi: 10.1038/s41467-022-30374-9

Figure Lengend Snippet: NIH-3T3 fibroblasts were treated with vehicle, TNFα (10 ng/ml), STS (0.3 µM), CHX (20 µg/ml), ETO (10 µM), TPG (2 µM), VAL (10 µM), MC (10 µM), or I3M (10 µM) or were serum starved (Serum) for the indicated period of time. a , d – f Cellular proportion of MUFAs in PI and PI(18:1/18:1) (left to right (LTR) P = 0.0000004, 0.00000007, 0.0000007) ( a ), PC (LTR P = 0.9999999986, 0.000002, 0.0000003) ( d ), PE (LTR P = 0.99991, 0.000000007, 0.99999992) ( e ), and PS (LTR P = 0.99992, 0.99998, 0.99997) ( f ); MUFAs: 16:1, 18:1. Data of ( a ), ( d ) is identical to w/o in Supplementary Figs. and . b Time-dependent changes of the cellular PI content. c Heatmap showing the time-dependent changes of the cellular PI profile ( P = 0.0007). Data are given as percentage of vehicle control for each time point. Mean ( c ) or mean + s.e.m. ( b ) and single data ( a , d – f ) from n = 3 ( a right panel, b , c , e , f ), n = 4 ( a left panel, d ) independent experiments. *** P < 0.001 for the respective time point ( b ) or P values given vs. vehicle control ( a , d – f ); repeated measures one-way ANOVA ( a , d – f ) of log data ( b ) + Tukey HSD post hoc tests.

Article Snippet: CHX, ETO, VAL, I3M, and CAY10566 were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques:

Journal: Nature Communications

Article Title: PI(18:1/18:1) is a SCD1-derived lipokine that limits stress signaling

doi: 10.1038/s41467-022-30374-9

Figure Lengend Snippet: Fibroblasts were cultivated under diverse cytotoxic conditions for 48 h or as indicated. a Negative correlation (−0.6 > r > −1) between cellular p-p38 MAPK (Thr180/Tyr182) levels (at 48 h) and the proportions of phospholipid (PL) species are shown for the co-regulated lipid network described in Supplementary Fig. . Correlations were calculated for mean p-p38 MAPK levels from three independent experiments. b Heatmap showing time-dependent changes in the activation of p38 MAPK compared to vehicle control for each time point. Representative Western blots are shown in Supplementary Fig. . STS, excluded due to pan-kinase inhibition; gray color for I3M, not determined. c , d Phosphorylation and expression of p38 MAPK ( P = 0.99995) ( c ) and JNK ( P = 0.000007) ( d ). Western blots are representative of five ( c ) or three ( b , d ) independent experiments. Data of c is identical to w/o in Supplementary Fig. , and . Mean ( b ) or mean + s.e.m. and single data ( c , d ) from n = 1 ( b I3M for 24 h), n = 2 ( b TNFα for 0.17 h, d for TPG), n = 3 ( a , b , d ), n = 4 ( b , c for CHX, I3M at 48 h), n = 5 ( b for 48 h, c ) independent experiments. P values given vs. vehicle control; mixed-effects model (REML) + Tukey HSD post hoc tests of log data ( c , d ). e Counter-regulation of PI(18:1/18:1) ratios and p38 MAPK activation during VAL-induced cell death across cell lines. MCF-7 breast adenocarcinoma cells, HEK293 embryonic kidney cells, primary human monocytes, MM6 acute monocytic leukemia cells, HT29 colon adenocarcinoma cells, HeLa cervical carcinoma cells, HepG2 hepatoma cells, and HUVECs were treated with vehicle or VAL (10 µM) for 48 h. Percentage changes in cellular PI(18:1/18:1) ratios and p-p38 MAPK levels were calculated vs. vehicle (100%), and the difference to the vehicle control is presented. Representative Western blots are shown in Supplementary Fig. . Detailed descriptions of datasets shown in panel e are given in Supplementary Note . P values given vs. vehicle control; two-tailed paired student t test.

Article Snippet: CHX, ETO, VAL, I3M, and CAY10566 were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Activation Assay, Western Blot, Inhibition, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: PI(18:1/18:1) is a SCD1-derived lipokine that limits stress signaling

doi: 10.1038/s41467-022-30374-9

Figure Lengend Snippet: Fibroblasts were cultivated under diverse cytotoxic conditions for 48 h ( a – c , e ) or as indicated ( d ). a Cellular proportion of non-esterified SFAs, MUFAs, and PUFAs. SFAs: 12:0, 14:0, 16:0, 18:0; MUFAs: 16:1, 18:1; PUFAs: 18:2, 20:4, 22:5, 22:6 (MUFA LTR P = 0.0146, 0.0128, 0.0023, 0.0155, 0.003; SFA LTR P = 0.0176, 0.0088, 0.0013, 0.0072, 0.0015). b Heatmap showing changes in the free fatty acid profile as compared to vehicle control. Data are given as percentage of the relative free fatty acid abundance. c Volcano plots highlighting free fatty acids that are strongly and significantly modulated by VAL or MC. Comparisons of the indicated treatment groups show the mean difference of percentage changes and the negative log10(adjusted P value). Adjusted P values given vs. vehicle control; two-tailed multiple unpaired student t tests from log data with correction for multiple comparisons using a two-stage linear step-up procedure by Benjamini, Krieger, and Yekutieli (false discovery rate 5%). d Heatmaps showing the time-dependent effect on Scd1 , Actb , and Gapdh mRNA levels that were normalized to the total amount of cellular RNA and compared to vehicle control for each time point. e Protein expression of SCD1. Western blots are representative of seven independent experiments (LTR P = 0.000000002, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0.\bar{99}$$\end{document} 0 . 99 ¯ ). Mean ( b – d ) or mean + s.e.m. ( a ) and single data ( e ) from n = 2 ( d for Scd1 and Gapdh at 6 h; Actb at 48 h for Serum), n = 3 ( a – d ), n = 6 ( e for TNFα, Serum, I3M), n = 7 ( e ) independent experiments. * P < 0.05, ** P < 0.01 or P values given vs. vehicle control; repeated measures one-way ANOVA ( a ) or mixed-effects model (REML) of log data ( e ) + Tukey HSD post hoc tests.

Article Snippet: CHX, ETO, VAL, I3M, and CAY10566 were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Two Tailed Test, Expressing, Western Blot