Strains harboring genetic circuit were streaked on the LB agar (2%) plate without antibiotics. E. coli MG1655 wild‐type and RPUG standard strains were streaked as controls. Individual colonies were picked from plates and were inoculated into M9 media without antibiotics. Cells were incubated overnight at 37°C and 1,000 rpm in an ELMI plate shaker using Nunc™ 96‐well plates (Thermo Scientific, USA, 249662). The next day, cells were then diluted 185‐fold into 200 μl of fresh M9 media without antibiotics and incubated for three hours at 37°C, 1,000 rpm in an ELMI plate shaker using Nunc™ 96‐well plates (Thermo Scientific, USA, 249662). After three hours, cells were 700‐fold diluted into M9 media without antibiotics and were induced with appropriate combinations of inducers such as 1 mM IPTG, 12.5 mM L‐arabinose, 20 ng/μl aTc, 10 μM OHC14, and 200 μM vanillic acid were used as indicated in Cello 2.0 prediction. After 5.5 h, 30 μl of cells was added to 200 μl 1× PBS with 2 mg/ml Kan for flow cytometry analysis. Median YFP fluorescence from each sample was analyzed using FlowJo (TreeStar, Inc., USA) software and was converted into RPUG.
Nunc 96 well plate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark, United Kingdom
The Nunc 96-well plates are a type of laboratory equipment used for various applications in cell culture, biochemical assays, and other experimental procedures. These plates have a standard 96-well format, with each well designed to hold a specific volume of liquid sample.
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Lab products found in correlation
Facsaria, by BD (3 mentions)
Magnetic microbead, by Miltenyi Biotec (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Rs4 11, by American Type Culture Collection (2 mentions)
Interleukin 7 (il 7), by R&D Systems (2 mentions)
Myelocult h5100, by STEMCELL (2 mentions)
Rpmi 1640 glutamax medium, by Thermo Fisher Scientific (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Trypsin edta 1x, by Thermo Fisher Scientific (2 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (2 mentions)
Fluostar omega, by BMG Labtech (2 mentions)
Biotek el808, by Agilent Technologies (2 mentions)
Victor3 1420 multilabel counter, by PerkinElmer (2 mentions)
Biotek synergy h1, by Agilent Technologies (1 mentions)
Opteia elisa set, by BD (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Prism 7, by GraphPad (1 mentions)
Sybrg 2, by Thermo Fisher Scientific (1 mentions)
Cd45 pe, by BioLegend (1 mentions)
Cd73 fitc, by BioLegend (1 mentions)
48 protocols using nunc 96 well plate
1
Measuring Genetic Circuit Performance
2
Characterizing NOT gate response functions
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Each strain containing a NOT gate was streaked on the LB agar (2%) plates with Kan (50 μg/ml) and Cm (35 μg/ml) antibiotics. A single colony was picked and inoculated into M9 media with Kan (50 μg/ml) and Cm (35 μg/ml) for overnight culture in an ELMI plate shaker at 37°C and 1,000 rpm. Cell cultures were performed using Nunc™ 96‐well plates (Thermo Scientific, USA, 249662). The next day, cells were 185‐fold diluted into 200 μl fresh M9 media with no antibiotics and incubated for 3 h in an ELMI plate shaker at 37°C and 1,000 rpm. After the 3 h, cells were then diluted again 700‐fold into 200 μl fresh M9 media with no antibiotics and were incubated with inducers for additional 5.5 h in an ELMI plate shaker at 37°C and 1,000 rpm. After the 5.5 h, 30 μl of cells was added to 200 μl 1× PBS with 2 mg/ml Kan for flow cytometry analysis. To measure OD, 150 μl aliquots were transferred to an optically transparent Nunc™ 96‐well plates (Thermo Scientific, USA, 165305) to measure OD600 using a Hybrid Microplate Reader BioTek Synergy H1 (BioTek Instruments Inc, USA). To measure the gate response functions, input and output promoter activities measured as median YFP fluorescence were converted into RPUG. The response functions were fit to a Hill equation y = ymin + ((ymax − ymin)/(1 + (x/K)n), using an in‐house Python script.
3
Cytokine Profiling in Lacrimal Glands
4
Culturing Patient-Derived Leukemia Cells
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Patient derived leukemia cells were cultured on MS5 stromal cells in the presence of 50 ng/mL recombinant human stem cell factor (SCF), 50 ng/mL recombinant human thrombopoietin (TPO), 10 ng/mL recombinant human FLT3 ligand (FLT3L), and 10 ng/mL recombinant human interleukin-7 (IL-7, all from R and D Systems) in Myelocult H5100 (Stem Cell Technologies) supplemented with 1% penicillin/streptomycin (Issaad et al., 1993 (link)) with modifications based on (Doulatov et al., 2010 (link)) to facilitate lymphoid cell growth; i.e., without horse serum or hydrocortisone (Issaad et al., 1993 (link)). Leukemia 1/MLL-AF4 was most amenable to in vitro culture and so was used for most culture-based experiments. For in vitro LDA experiments, 5,000 MS5 cells were plated in wells of gelatin-coated Nunc 96 well plates (Fisher Scientific) with cytokines 48 hours prior to FACS-based sorting of leukemia cells directly into the wells.
Human MV4; 11 and RS4; 11 cells (ATCC) were cultured in RPMI with 10% fetal calf serum supplemented with penicillin and streptomycin.
As indicated inTable S1 , some specimens were subjected to focused somatic mutation analysis (Rapid Heme Panel (Kluk et al., 2016 (link))).
Human MV4; 11 and RS4; 11 cells (ATCC) were cultured in RPMI with 10% fetal calf serum supplemented with penicillin and streptomycin.
As indicated in
5
Culturing Patient-Derived Leukemia Cells
6
HBV Epsilon RNA Binding Assay
7
Osteogenic Differentiation of MSC Subpopulations
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AD-MSC lines were washed with PBS and stained with ALP-APC (R&D) (1/50) and CD73-FITC (Biolegend) (1/160) for 25 min at 4 °C. Upon washing, the cell fractions (controls sorted, ALP+/CD73+, ALP−/CD73high, ALP−/CD73low) were sorted with a FACS BD Aria III 5L and seeded in Nunc™ 96-well plates (TPP) at a density of 1.2 × 104 cells/cm2 for osteogenic differentiation. Controls sorted were unstained cells processed through the FACS and collected without sorting specific subpopulations. Differentiation was induced 24 h after seeding. Freshly isolated SVFs were washed with PBS and stained with ALP-APC (R&D) (1/50), CD73-FITC (Biolegend) (1/160), and CD45-PE (Biolegend) (1/160) for 25 min at 4 °C. SVF fractions (controls sorted, CD45−/ALP+/CD73+, CD45−/ALP−/CD73high, CD45−/ALP−/CD73low) were sorted with FACS BD Aria III 5L and plated in vitro at a density of 1 × 104 in 96-well plates (TPP) for osteogenic differentiation. All media were changed every 4 days.
8
Cytotoxicity Assay with Jurkat Cells
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Target cells and effector Jurkat cells were seeded into Nunc™ 96-well plates (Nunc, # 168136) at an effector to target cell (E:T) ratio of 5:1. The 3T3 cell line was used as negative control while EGFR-expressing 3T3 EGFR stable transfected cells were used as the positive control. Antibody test samples diluted in assay media (supplemented RPMI-1640 Glutamax medium; Gibco, # 41966-029) were added into appropriate wells. After an overnight co-culture period at 37°C, the plates were centrifuged and cell supernatants were collected for ELISA analysis. The effector cells were harvested and stained with anti-CD69 FITC antibody, diluted 1:100 (Biolegend, #104506), and anti-HLA class I APC antibody, diluted 1:100 (Biolegend, # 311410) for 30 min at 4°C. Cells were washed twice in PBS + 1% FBS and resuspended in 200 μL of 7-AAD viability dye diluted 1:200 (Invitrogen, #A1310). Samples were run on a Novocyte flow cytometer (ACEA Biosc Inc.). Data was analysed using FlowJo version 10 software (FlowJo, LLC).
9
Antibody-Dependent Cell Cytotoxicity Assay
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Target cells were seeded overnight into Nunc™ 96-well plates (Nunc, #161093). A431, HT-29 and MCF7 cells lines, which express the targeted antigen EGFR, were used as positive cell lines while a CHO cell line was used as a negative control cell line. After 24 h of incubation, antibody test samples diluted in assay media (supplemented RPMI-1640 Glutamax medium; Gibco, # 32404-014) and freshly isolated effector cells PBMC (E:T = 5:1) were added into appropriate wells. After 48 h of co-culture, a final concentration of 1% Triton X-100 (Sigma-Aldrich, #T8787) solution was added into high control wells to obtain maximal cell lysis. Target cells incubated with effector PBMC only were used as the low control. The plates were centrifuged and cell supernatants were collected for cytotoxicity analysis using the LDH cytotoxicity detection kit (Takarabio, #MK401) following the manufacturer’s protocol. 100 µL of cell supernatant was added to 100 µL of reaction mixture, which was prepared according to the manual and incubated for 10–15 min at RT. Absorbance was measured at 492 nm (600 nm as reference wavelength) on a Clariostar plate reader (BMG Labtech). Data was analysed in GraphPad Prism v. 8. Cytotoxicity was calculated following the equation, Cytotoxicity (%) = ((Experiment value − Low control) / (High control − Low control)) x 100.
10
Pituitary Dissociation and Immune Depletion
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Whole pituitaries were dissected from wild-type C57BL/6 female mice at 9–10 weeks of age. Whole pituitaries were isolated into ice-cold PBS and then dispersed by incubation with 0.25% collagenase Type IV and 0.25% trypsin–EDTA (1x) (Life Technologies) as previously described (23 (link)). For immune depletion studies, dispersed pituitary was divided in half, with one half being subjected to depletion of total CD45+ immune cells using Miltenyi Biotech anti-C45 micro beads according to protocol. The cells (dispersed pituitary or immune cell depleted pituitary) were plated on poly-l-lysine (Sigma-Aldrich Inc.) coated Nunc 96-well plates (Thermo Fisher Scientific) at a density of 1.5 × 106 cells per cm2. The cells were cultured for 24 h at 37°C and 5% CO2 in high-glucose HEPES-buffered DMEM with 10% FBS prior to experimentation. After pituitary cultures equilibrated they were treated serum starved for 16 hrs, followed by a change in media and 30 min treatment with or without GnRH.
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