microfluidics device with flow control y04c cellasic plate with onix controllers  (CellASIC Corporation)

Journal: bioRxiv

Article Title: Deep learning-driven imaging of cell division and cell growth across an entire eukaryotic life cycle

doi: 10.1101/2024.04.25.591211

Figure Lengend Snippet: (A) Schematic of S. cerevisiae life cycle. F0 = parental generation, F1 = filial generation 1 (B) Schematic microfluidic protocol to specifically trigger S. cerevisiae’s sexual life cycle by selecting against non-sporulated cells that can outcompete germinating spores. Nuclei represented as solid shapes inside the cells. (C-D) Representative time lapse micrographs of computationally aligned yeast cells undergoing (C) homothallic life cycle type A, in which sporulation is followed by ascus mating directly leading to two diploid cells without an intervening proliferative haploid state, or (D) Homothallic life cycle type B, in which sporulation is followed by partial ascus mating producing one diploid and two proliferative haploid cells which eventually diploidize through inbreeding.

Article Snippet: The culture was spun down on a tabletop microfuge for 3 seconds to remove clumps, and 70 μl of the top layer were transferred to a microfluidics device with flow control (Y04C CellASIC plate with OniX controllers), pre-warmed at 25 °C.

Techniques:

Journal: bioRxiv

Article Title: Deep learning-driven imaging of cell division and cell growth across an entire eukaryotic life cycle

doi: 10.1101/2024.04.25.591211

Figure Lengend Snippet: (A) Schematic microfluidic induction of sequential S. cerevisiae sexual life cycles. Solid circles = nuclei; green = LiCH, orange = Whi5-mSC. (B) Representative scaled average size, Whi5-mSC or LiCHI nuclear concentration during the transition from cell birth into the first mitotic division aligned to the peak of Whi5 concentration during G1 (C-E) Boxplot comparison of the first mitotic division in three sexually reproducing generations (F0-F2) in terms of (C) cell size, (D) peak nuclear Whi5 levels, (E) total Whi5 cell concentration. (F-H) Boxplot comparison of the kinetics of meiotic divisions in three sexually reproducing generations (F0-F2) in terms of the duration between the time point of anaphase I and (F) pre-meiotic G1 exit, (G) prophase I exit, (H) meiotic exit. (I-K) Correlation between the time of anaphase I and pre-meiotic G1 exit in (I) F0, (J) F1, and (K) F2 generations. (L-N) Correlation between the time of anaphase I and cell size at anaphase I (L) F0, (M) F1, and (N) F2 generations.

Article Snippet: The culture was spun down on a tabletop microfuge for 3 seconds to remove clumps, and 70 μl of the top layer were transferred to a microfluidics device with flow control (Y04C CellASIC plate with OniX controllers), pre-warmed at 25 °C.

Techniques: Concentration Assay, Comparison

Journal: mBio

Article Title: Polar Growth in Corynebacterium glutamicum Has a Flexible Cell Wall Synthase Requirement

doi: 10.1128/mBio.00682-21

Figure Lengend Snippet: RodA and PBP1b promote cell growth by inserting nascent PG material at the poles. Overnight cultures of the indicated strains from Fig. 1 grown in BHIS medium at 30°C were diluted 1:1,000 in BHIS and grown for 3 h at 30°C. The cells were loaded into a CellASIC microfluidic device and grown for 30 min in BHIS medium at 30°C. Following this equilibration period, the cells were imaged every 5 min using phase-contrast and fluorescence optics. Cells were pulse labeled with the fluorescent d -amino acid TADA for 3 min at the 6-min mark. The label was then progressively washed away by the flow of fresh medium lacking label. Every 6th frame in the time-lapse series is shown. The length of unlabeled cell wall was measured after the TADA pulse ( t = 20 min) and at the end of the time lapse ( t = 120 min) using Oufti ( N > 150 cells); the unlabeled portion of each cell was defined as that with a TADA signal <20% of the maximum TADA signal for that cell. The mean and standard error of the rate of polar elongation were calculated by taking the mean difference in the length of unlabeled cell wall at the initial and final time points divided by the time elapsed.

Article Snippet: For experiments that utilized the CellASICs device, cells were loaded into the CellASIC Onix B04 microfluidic plates (Millipore Sigma) that were attached to the microscope by using a multiwell insert.

Techniques: Fluorescence, Labeling

Journal: mBio

Article Title: Polar Growth in Corynebacterium glutamicum Has a Flexible Cell Wall Synthase Requirement

doi: 10.1128/mBio.00682-21

Figure Lengend Snippet: Spatial distribution of RodA and the aPBPs at the cell poles. (A) Representative fluorescence micrographs of Cglu cells producing the indicated mScar fusions as the sole copy of the corresponding genes either from the native locus (DivIVA-mScar, strain HL23) or integrated plasmids pJSW19 (mScar-PBP1a) in strain HL37 (Δ ponA ), pJSW18 (mScar-CofA) in strain JS8 (Δ cofA ), pJWS94 (mScar-PBP1b) in strain JS20 (Δ ponB ), or pJWS33 (mScar-RodA) in strain HL31 (Δ rodA ). Overnight cultures grown in BHI medium at 30°C were diluted 1:1,000 in BHI medium supplemented with 0.3 mM theophylline and grown at 30°C. When the OD 600 reached 0.2 to 0.3, cells were diluted 10-fold and loaded into a CellASIC microfluidic device for imaging by fluorescence microscopy. (B) Quantification of polar fluorescence distributions of the indicated mScar fusions. Following cell segmentation by Oufti , a MATLAB-based script was used to identify the brightest pole of each cell, aligning the cells by setting the tip of the brightest pole to position zero. The average fluorescence intensity distribution across all cells as a function of distance from the pole was then quantified. The fluorescence profiles shown were plotted following background subtraction.

Article Snippet: For experiments that utilized the CellASICs device, cells were loaded into the CellASIC Onix B04 microfluidic plates (Millipore Sigma) that were attached to the microscope by using a multiwell insert.

Techniques: Fluorescence, Imaging, Microscopy