Journal: STAR Protocols

Article Title: Large-scale single-cell cloning of genome-edited cultured human cells by On-chip SPiS

doi: 10.1016/j.xpro.2023.102364

Figure Lengend Snippet:

Article Snippet: SAPPHIRE LOW-RETENTION PIPETTE TIPS , Greiner Bio-One , Cat# 738259.

Techniques: Recombinant, Ligation, Modification, Knock-Out, Extraction, Transfection, Membrane, Plasmid Preparation, Sequencing, Software, Transferring, Sterility

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: Current issues in molecular biology

Article Title: A Group of Tumor-Suppressive micro-RNAs Changes Expression Coordinately in Colon Cancer.

doi: 10.3390/cimb45020063

Figure Lengend Snippet: Figure 2. Correlation matrices between microRNAs in normal (A) and tumoral (B) tissue; the values on the color scale represent the Spearman ρ coefficient. (C,D) Correlation network in normal and tumoral tissue, respectively. Continuous line- correlations with Spearman ρ coefficient ≥0.5; dashed line-significant correlations with Spearman ρ coefficient < 0.5; green line-correlations in healthy tissue; red line-correlations in the tumoral tissue. The molecules had significant correlations between them, stronger in the tumoral tissue.

Article Snippet: A total of 50 ng RNA for miRNA (miR-29a-5p, miR-146a-5p, miR 215-5p, miR 449-5p) expression was transcribed into cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA).

Techniques:

Journal: Current issues in molecular biology

Article Title: A Group of Tumor-Suppressive micro-RNAs Changes Expression Coordinately in Colon Cancer.

doi: 10.3390/cimb45020063

Figure Lengend Snippet: Figure 3. Transcription factors that regulate the four miRNAs. (A) miR-146A; (B) miR-29A; (C) miR- 215; (D) miR-449. MiR-micro-RNA; Levels 1,2- FT-miRNA regulations derived from ChIP sequencing; level 2 promoter-supported by high-throughput experimental data; TF-transcription factor; mi-RNA- micro-RNA; (TransMir, v.2.0) [31].

Article Snippet: A total of 50 ng RNA for miRNA (miR-29a-5p, miR-146a-5p, miR 215-5p, miR 449-5p) expression was transcribed into cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA).

Techniques: Derivative Assay, ChIP-sequencing, High Throughput Screening Assay

Journal: Current issues in molecular biology

Article Title: A Group of Tumor-Suppressive micro-RNAs Changes Expression Coordinately in Colon Cancer.

doi: 10.3390/cimb45020063

Figure Lengend Snippet: Figure 4. The correlation network between the transciption factors that regulate the four miRNAs (GeneMania and Cytoscape GeneMania plug-in) [32,33].

Article Snippet: A total of 50 ng RNA for miRNA (miR-29a-5p, miR-146a-5p, miR 215-5p, miR 449-5p) expression was transcribed into cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA).

Techniques:

Journal: Current issues in molecular biology

Article Title: A Group of Tumor-Suppressive micro-RNAs Changes Expression Coordinately in Colon Cancer.

doi: 10.3390/cimb45020063

Figure Lengend Snippet: Figure 5. Functional analysis of the correlation network between the TFs that control the expression of the miRNAs. (A) Network with complex nodes (Cytoscape with EnrichmentMap plug-in) [33,34], Red dots-network nodes; Blue lines-network edges. (B) The diagram highlights functional modules in the network (Cytoscape with Reactome FI plug-in) [35]: (C) correlations between the network modules (Cytoscape with AutoAnnotate plug-in) [33]. TF-transcription factor; EMT-epithelial-to-mesenchimal transition; G1,S,G2,M-stages in cell cycle; UPR- unfolded protein response; RTK-receptor-thyrosin-kinase.

Article Snippet: A total of 50 ng RNA for miRNA (miR-29a-5p, miR-146a-5p, miR 215-5p, miR 449-5p) expression was transcribed into cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA).

Techniques: Functional Assay, Control, Expressing

Journal: Current issues in molecular biology

Article Title: A Group of Tumor-Suppressive micro-RNAs Changes Expression Coordinately in Colon Cancer.

doi: 10.3390/cimb45020063

Figure Lengend Snippet: Figure 6. Proposed model concerning the cellular networks and pathways that control the expression of the four miRNAs. It can be seen that in the case of cell cycle-related processes, there is a stream of events that begins with the signaling pathways activation, then the immediate-early genes are activated, then the transition G0-G1 occurs, followed by the transition G1-S and G2-M, and finally, the mitotic exit and the differentiation of the daughter cells take place. The successive transcriptional waves are coordinated by specific master TFs for each one of them, but each step is accomplished through the action on the chromatin, which makes possible the basic transcription; after that, the post- transcriptional regulation through miRNAs occurs (all these processes were shown to be correlated in our analysis). The differentiation is signaled from the start through the presented pathways, but it occurs only at the end of the cell cycle (when also signaling events may occur). It has also been shown to correlate with these basic chromatin, transcription processes and regulation through miRNAs. The other processes (response to stimuli, response to hormones, transcription in response to stress) occur independently, (in response to their own stimuli), but in networks that communicate with the cell cycle. Red, light brown or yellow lines- correlations from the present study; green line- miRNA circuits; UPR-Unfolded protein response (Endoplasmic reticulum stress); TF-transcription factors; G1,S,G2,M- stages in the cell cycle. The model was built based on the conclusion of the present study and also on other literature data [10,14,20,24,36,37].

Article Snippet: A total of 50 ng RNA for miRNA (miR-29a-5p, miR-146a-5p, miR 215-5p, miR 449-5p) expression was transcribed into cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA).

Techniques: Control, Expressing, Protein-Protein interactions, Activation Assay