Journal: eLife

Article Title: Robotic search for optimal cell culture in regenerative medicine

doi: 10.7554/eLife.77007

Figure Lengend Snippet: ( A ) Schematic diagram of the standard iPSC-RPE differentiation procedures. DDay indicates the differentiation day. Filled circles represent days when the robot operated, solid circles represent days with human operations only, and dashed line circles represent days when no operations were conducted. F stands for FGF receptor inhibitor; Y for Y-27632, a Rho-kinase inhibitor; SB for SB431542, a TGF-β/Activin/Nodal signal inhibitor; CKI for a CKI-7, Wnt signal inhibitor; and MX for medium exchange. ( B ) The LabDroid Maholo including peripheral equipment. ( C ) Plate numbering and the orders of seeding, passage, and medium exchange operations. Eight 6-well plates were used for each experiment. ( D ) Well numbering. ( E ) Scores of the first trial. iPSC-RPE differentiation was conducted under six different trypsin treatment times using the LabDroid. Yellow bars represent the pigmented cell area score of each well. The bold black lines and the shaded area around the lines represent the mean score and SEM of eight samples operated at the same trypsin time, respectively. The raw values are shown in . Figure 2—source data 1. Acquired pigmented images of the baseline experiment. Images acquired on Day 34 of the baseline experiment; images of the bottom of the well with cultured cells, cropped to the size of the well. These 8-bit images were adjusted to a minimum and maximum contrast value of 100 and 150, respectively. IDs on the bottom indicate 'B (baseline) - Plate No. - Well No.'. Figure 2—source data 2. Executed parameters and scores of the baseline experiment. Related to . Raw values of the parameter candidates and pigmentation scores in the baseline experiment. *KSR concentration was lowered in a systematic fashion, unlike the KP parameter. Specific values: DDays 1–3, 20% KSR; DDays 4–7, 15% KSR; from DDay 8, 10% KSR. Figure 2—source data 3. Pipetting volume and pipette combination. Related to . Given the limitations of the LabDroid, setting the micropipette to an arbitrary volume was difficult. Therefore, we pseudo-implemented a fine volume setting for the transfer of 0–1000 µL by combining nine micropipettes with pre-set volumes (3000, 1000, 450, 300, 200, 80, 30, 10, and 5 µL). The number of combinations was limited to three or fewer. The numbers in the table indicate the micropipettes and the number of times they had to be used to achieve the desired volumes. For example, 260 µL indicates that the 200 and 30 µL micropipettes had to be used once and twice, respectively.

Article Snippet: For human use: micropipette tip, 2140-05-HR/2149P-05/61849, Thermo Fisher Scientific Inc (MA, USA); micropipette tip, 30389165, Mettler Toledo (OH, USA); micropipette tip, 737251, Greiner Bio-One International GmbH (Germany); disposable pipette, 356507, Corning Incorporated (NY, USA); disposable pipette, 606160/607160/760160/768160, Greiner Bio-One International GmbH (Germany); filtration, SLGVJ13SL, Merck & Co., Inc (NJ, USA); filtration, SS-10LZ, Terumo Corporation (Japan); filtration, 431096/430281/431097/430282, Corning Incorporated (NY, USA); 1.5 mL tube, 72.692MS, Sarstedt K.K. (Japan); 15 mL tube, 352096, Corning Incorporated (NY, USA); 50 mL tube, 352070, Corning Incorporated (NY, USA).

Techniques: Cell Culture, Concentration Assay, Transferring

Journal: eLife

Article Title: Robotic search for optimal cell culture in regenerative medicine

doi: 10.7554/eLife.77007

Figure Lengend Snippet: Robotic experiments were conducted for 185 days, with a total robot operating time of 995 hr, 39 min, and 21 s . Each round consisted of 73 jobs, and five rounds were performed, including baseline and validation. The total number of jobs executed by the LabDroid was 365, of which 343 were successful on the first try; 22 required human intervention at least once. The job success rate was 93.973%. One job consisted of multiple commands. The total number of commands that the LabDroid was ordered to execute was 75039, of which 75,011 were successful on the first try, 25 required human intervention at least once, and 3 were aborted. The command success rate was 99.963%. The reasons for the errors included micropipette tip loading error, 4 commands; micropipette tip ejection error, 7 commands; microscope and its control PC-derived errors, 13 commands (including 3 aborted commands); defective labware, 1 command; and human error, 3 commands. The errors occurred on the following dates: 2019 Feb (baseline), 11 commands; 2019 Apr (round 1), 11 (including three aborted commands); 2019 Jul (round 2), 4; 2020 Jan (round 3), 0; and 2020 Mar (validation), 2. The number of motions requiring the use of micropipettes that the LabDroid was ordered to execute was 39421; the rate of failure of either tip loading or ejection was 0.0279%. The vertical and horizontal axes represent the causes and error timing of the errors that occurred during the robotic experiments, respectively. Each circle represents one error.

Article Snippet: For human use: micropipette tip, 2140-05-HR/2149P-05/61849, Thermo Fisher Scientific Inc (MA, USA); micropipette tip, 30389165, Mettler Toledo (OH, USA); micropipette tip, 737251, Greiner Bio-One International GmbH (Germany); disposable pipette, 356507, Corning Incorporated (NY, USA); disposable pipette, 606160/607160/760160/768160, Greiner Bio-One International GmbH (Germany); filtration, SLGVJ13SL, Merck & Co., Inc (NJ, USA); filtration, SS-10LZ, Terumo Corporation (Japan); filtration, 431096/430281/431097/430282, Corning Incorporated (NY, USA); 1.5 mL tube, 72.692MS, Sarstedt K.K. (Japan); 15 mL tube, 352096, Corning Incorporated (NY, USA); 50 mL tube, 352070, Corning Incorporated (NY, USA).

Techniques: Biomarker Discovery, Microscopy, Control, Derivative Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Anatomically Resolved Mouse Brain Proteome Reveals Parkinson Disease-relevant Pathways *

doi: 10.1074/mcp.M116.061440

Figure Lengend Snippet: Sample preparation for regional analysis of proteins. A, 17 surgically distinctive mouse brain regions were isolated using 1-mm inner diameter punches from coronal section. B, simplified sample preparation method. Mouse brain regional samples were isolated from 1-mm-thick coronal sections followed by in-solution digestion. sRP-RP was used for separate and concentrate-digested peptide. Orbitrap instrument was used for MS analysis. First dimensional separation is done with high pH reverse phase micro-column using C18 beads. Stepwise ACN gradient was applied, and eluent was pooled into six fractions depending on ACN concentration before MS/MS. C, different continuous ACN gradient applied for different high pH eluent pools based on hydrophobicity nature of each pools.

Article Snippet: A micro-pipette tip C18 column was made from a 200-μl pipette tip by layering 6 mg of C18 matrix (Reprosil-Pur Basic C18, 3 μm, Dr. Maisch GmbH, Germany) on top of the C18 disk (3M, Empore TM C18) plug.

Techniques: Sample Prep, Isolation, Concentration Assay, Tandem Mass Spectroscopy