HEK293T cells were seeded into 96-well flat-bottom cell culture plates (Corning) for PE treatment at 1.2 × 104 cells/well. Transfections were carried out 18–24 h post-seeding with 30 ng PE2 plasmid, 10 ng pegRNA, and 3.3 ng ngRNA plasmid per transfection (per well, in a 96-well plate). TransIT-X2 (Mirus) was used as the lipofection reagent at 0.3 μL per transfection.
96 well flat bottom cell culture plate
Manufactured by Corning
Sourced in United States
The 96-well flat-bottom cell culture plates are a standard laboratory equipment used for cell-based experiments. These plates provide a flat, uniform surface for culturing cells in a 96-well format, allowing for multiple samples to be tested simultaneously. The plates are designed to facilitate efficient cell growth, attachment, and monitoring in a controlled environment.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Se cell line nucleofector x kit, by Lonza (2 mentions)
Sf cell line nucleofector x kit, by Lonza (2 mentions)
Se cell line 4d nucleofector x kit, by Lonza (2 mentions)
24 well cell culture plate, by Corning (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Corning (2 mentions)
Glutamax, by Corning (2 mentions)
48 well poly d lysine coated plates, by Corning (2 mentions)
Dmem plus glutamax, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Le ma900 cell sorter, by Sony (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
35 μm cell strainer, by Corning (2 mentions)
Hi fbs, by Thermo Fisher Scientific (1 mentions)
L glutamine, by American Type Culture Collection (1 mentions)
Bj fibroblasts, by American Type Culture Collection (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
Cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
13 protocols using 96 well flat bottom cell culture plate
1
CRISPR-Based Gene Editing in HEK293T Cells
2
Efficient Transfection and Genomic Editing
3
Efficient Transfection and Genomic Editing
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HEK293T cells were seeded at 1.25 × 104 cells per well into 96-well flat bottom cell culture plates (Corning) for DNA on-target experiments or at 6.25 × 104 cells per well into 24-well cell culture plates (Corning) for DNA off-target experiments. 24 hours post-seeding, cells were transfected with 30 ng of control or base/prime editor plasmid and 10 ng of peg or gRNA plasmid (and 3.3 ng nicking gRNA plasmid for PE3) using 0.3 μL of TransIT-X2 (Mirus) lipofection reagent for experiments in 96-well plates, or 150 ng control or base editor plasmid and 50 ng gRNA, and 1.5 μL TransIT-X2 for experiments in 24-well plates. K562 cells were electroporated using the SF Cell Line Nucleofector X Kit (Lonza), according to the manufacturer’s protocol with 2 × 105 cells per nucleofection and 800 ng control or base/prime editor plasmid, 200 ng gRNA or pegRNA plasmid, and 83 ng nicking gRNA plasmid (for PE3). U2OS cells were electroporated using the SE Cell Line Nucleofector X Kit (Lonza) with 2 × 105 cells and 800 ng control or base/prime editor plasmid, 200 ng gRNA or pegRNA, and 83 ng nicking gRNA (for PE3). HeLa cells were electroporated using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 5 × 105 cells and 800 ng control or base/prime editor, 200 ng gRNA or pegRNA, and 83 ng nicking gRNA (for PE3). 72 hours post-transfection, cells were lysed for extraction of genomic DNA (gDNA).
4
BJ Fibroblast mRNA Transfection Protocol
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BJ fibroblasts from the American Type Culture Collection (ATCC; Manassas, VA) were cultured in EMEM (Eagle’s minimum essential medium) growth media with l -glutamine (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies, Waltham, MA, USA). Cells were seeded in 96-well flat-bottom cell culture plates (Corning Inc., Corning, NY, USA) at 20,000 cells per well for 24 hours before transfection. Cells were transfected with mRNA (250 ng per well) using Lipofectamine 2000 (Thermo Fisher Scientific). Cell culture supernatants were harvested 48 hours after transfection for analyses.
5
CRISPR-Mediated HBB E6V Mutation Generation
6
CRISPR-Mediated HBB E6V Mutation Generation
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2.5 × 104 HEK293T cells were seeded on 48-well
poly-D-lysine coated plates (Corning). 16-24 h post-seeding, cells were
transfected at approximately 70% confluency with 1 μL of Lipofectamine
2000 (Thermo Fisher Scientific) according to the manufacturer’s protocols
and 750 ng of PE2-P2A-GFP plasmid, 250 ng of pegRNA plasmid, and 83 ng of sgRNA
plasmid. After 3 days, cells were washed with 1× PBS (Gibco) and
dissociated using TrypLE Express (Gibco). Cells were then diluted with DMEM plus
GlutaMax (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Gibco) and
passed through a 35-μm cell strainer (Corning) prior to sorting. Flow
cytometry was carried out on a LE-MA900 cell sorter (Sony). Cells were treated
with 3 nM DAPI (BioLegend) 15 minutes prior to sorting. After gating for doublet
exclusion, single DAPI-negative cells with GFP fluorescence above that of a
GFP-negative control cell population were sorted into 96-well flat-bottom cell
culture plates (Corning) filled with pre-chilled DMEM with GlutaMax supplemented
with 10% FBS. SeeSupplementary Note 1 for representative FACS sorting examples and
allele tables. Cells were cultured for 10 days prior to genomic DNA extraction
and characterization by HTS, as described above. A total of six clonal cell
lines were identified that are homozygous for the E6V mutation in
HBB.
poly-D-lysine coated plates (Corning). 16-24 h post-seeding, cells were
transfected at approximately 70% confluency with 1 μL of Lipofectamine
2000 (Thermo Fisher Scientific) according to the manufacturer’s protocols
and 750 ng of PE2-P2A-GFP plasmid, 250 ng of pegRNA plasmid, and 83 ng of sgRNA
plasmid. After 3 days, cells were washed with 1× PBS (Gibco) and
dissociated using TrypLE Express (Gibco). Cells were then diluted with DMEM plus
GlutaMax (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Gibco) and
passed through a 35-μm cell strainer (Corning) prior to sorting. Flow
cytometry was carried out on a LE-MA900 cell sorter (Sony). Cells were treated
with 3 nM DAPI (BioLegend) 15 minutes prior to sorting. After gating for doublet
exclusion, single DAPI-negative cells with GFP fluorescence above that of a
GFP-negative control cell population were sorted into 96-well flat-bottom cell
culture plates (Corning) filled with pre-chilled DMEM with GlutaMax supplemented
with 10% FBS. See
allele tables. Cells were cultured for 10 days prior to genomic DNA extraction
and characterization by HTS, as described above. A total of six clonal cell
lines were identified that are homozygous for the E6V mutation in
HBB.
7
Cytokine profiling of activated PBMCs from EPT infants
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In this study, PBMCs from 107 EPT infants were used for stimulation. Frozen PBMCs were thawed gently and washed with RPMI‐1640 supplemented with 20 mm HEPES (GE Healthcare Life Sciences). The cells were counted and checked for viability with Trypan Blue staining. Subsequently, the cells were resuspended in a concentration of 106 cells mL−1 in a cell culture medium, consisting of RPMI‐1640 supplemented with 20 mm HEPES, 100 U mL−1 penicillin, 100 ug mL−1 streptomycin, 2 mm L‐glutamate (2 mm ; all GE Healthcare Life Sciences) and 10% heat‐inactivated FCS (Sigma‐Aldrich). The cells were then harvested in 96‐well flat bottom cell culture plates (Corning Incorporated, Corning, NY) at a concentration of 5 × 105 cells in 200 μL volume per well followed by stimulation with the human T‐cell activator CD3/CD28 beads (Gibco by Life Technologies) at 2:1 (cell:bead) ratio or kept unstimulated. Cells were incubated for 24 h at 37°C and 5% CO2. After 24 h of incubation, supernatants were collected, centrifuged and stored at −70°C for later cytokine and chemokine analyses.
8
Assessing MEK Inhibitors on MDA-MB-231 Cells
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MDA-MB-231 cells were plated at a density of 1 × 104 cells in 96-well flat-bottom cell culture plates (Corning). The next day, the cells were starved for 4 h in RPMI medium only before the addition of 10 nM or 1 μM of the MEK1/2 inhibitor RDEA119 (Cat: 1089), the MEK5 inhibitor BIX02189 (Cat: S1531) (both from Selleckchem) or a similar volume of DMSO for 24–48 h. The medium was collected for G-CSF ELISA analysis after 24 and 48 h and replaced by 100 μL of RPMI containing 10 μL of the CCK8 reagent (Cat: CK04–11, Dojindo) for cell viability analysis. All experiments were performed in triplicates with 3–4 repetitions. The G-CSF level for each well was normalized to the corresponding absorbance signal.
9
Basophil Degranulation Assay for Antigen Responses
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Rat basophil leukaemia cells (RBL-2H3) grown in supplemented RPMI 1640 medium (Gibco, Bleiswijk, The Netherlands) were seeded at 4 x 105 cells/well into 96-well flat bottom cell culture plate (Corning incorporated, Kennebank, OH) and cultured overnight at 37°C in 5% CO2. Cells were then incubated with 1:10 dilutions of a serum pool generated from each mouse-group in triplicates at 37°C and 5% CO2 for 2 hours. Thereafter, supernatants were removed and cells were washed twice with Tyrode’s buffer/0.1% BSA (Sigma-Aldrich, UK). IgE-loaded cells were then stimulated with different concentrations of antigens at 37°C for 30 minutes and mediator release was detected in the cell supernatants with the addition of 4-methylumbelliferyl β-D-galactopyranoside (4-MUG, Sigma Aldrich). For determination of 100% mediator release, cells were lysed with 10% v/v Triton X-100 (Merck Millipore, Darmstadt, Germany). The fluorescence of β-hexosaminidase release was measured between wavelengths of 360nm to 465nm using an Infinite 200 PRO microplate reader (Tecan, Maennedorf, Switzerland). The results are calculated as the percentages of total β-hexosaminidase released with the addition of 10% triton.
10
Cytotoxicity Evaluation of Magnetic Nanoparticles
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The cytotoxicity of MNPs in DECs and DMCs was assessed by MTS assays. In brief, DECs or DMCs were seeded onto a 96-well flat bottom cell culture plate (Corning Inc., Corning, NY, USA) at 3 × 103 cells/well and incubated with MNPs at 0, 50, 100, 150, or 300 pg-magnetite/cell. After 24 h of incubation at 37 °C, the cells were exposed to an MTS solution for 24 h. Suspended cells were removed by gentle rinsing with phosphate-buffered saline (PBS), and the number of adherent cells remaining in each well was quantified using a coupled enzymatic assay resulting in conversion of a tetrazolium salt into a red formazan product (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega, Madison, WI, USA). Recording the absorbance at 490 nm in the MTS assay was carried out using a microplate reader (infinite M200, Tecan Japan Co., Ltd., Kanagawa, Japan).
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