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Biocoat plates

Manufactured by Corning
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BioCoat plates are cell culture products designed for various cell-based applications. They feature a surface treatment that promotes cell attachment and growth. The plates are available in standard cell culture formats and can be used with a range of cell types.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (26 mentions) 40 μm strainer, by Thermo Fisher Scientific (2 mentions) Irfp670, by Addgene (2 mentions) Moflo xdp cell sorter, by Beckman Coulter (2 mentions) 4d nucleofector, by Lonza (2 mentions) Jc 1 dye, by Merck Group (2 mentions) Infinite m1000 pro, by Tecan (2 mentions) Proteinase k, by Thermo Fisher Scientific (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Se cell line 4d nucleofector x kit s, by Lonza (1 mentions) Human fibronectin, by Corning (1 mentions) Vancomycin, by Merck Group (1 mentions) Ceftazidime, by Merck Group (1 mentions) Penicillin streptomycin amphotericin b, by Corning (1 mentions) Collagen coated, by Corning (1 mentions) M per buffer, by Thermo Fisher Scientific (1 mentions) Rnasy plus 96 kit, by Qiagen (1 mentions)

17 protocols using biocoat plates

1

Efficient Transfection and Sorting of Diverse Cell Lines

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HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. Briefly, 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (ThermoFisher Scientific) per well according to the manufacturer's protocol.
Astrocytes, U2OS, HCC1954 and HEK293T cells were transfected using appropriate Amaxa Nucleofector™ II programs according to manufacturer's instructions (basic glial cell, V, V, and V kits using programs T-020, X-001, X-005, and Q-001 for astrocytes, U2OS, HCC1954, and HEK293T cells, respectively). 40 ng of iRFP670 (Addgene plasmid 45457)33 was added to the nucleofection solution to assess nucleofection efficiencies in these cell lines. Astrocytes and HCC1954 cells were filtered through a 40 μm strainer (Fisher Scientific) after harvesting, and the nucleofected cells were collected on a Beckman Coulter MoFlo XDP Cell Sorter using the iRFP signal (abs 643 nm, em 670 nm). The U2OS and HEK293T cells were used without enrichment of nucleofected cells.
Komor A.C., Kim Y.B., Packer M.S., Zuris J.A, & Liu D.R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420-424.
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2

CRISPR-Mediated Gene Editing and Activation in HEK293T Cells

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HEK293T cells were seeded on 48-well poly-D-lysine-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. For genomic DNA cutting or base editing, 750 ng of Cas9 or BE3 and 250 ng of sgRNA expression plasmids were transfected using 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturer’s protocol. For GFP activation, 200 ng of dCas9–VPR plasmid, 50 ng of sgRNA expression plasmid, 60 ng of GFP reporter plasmid, and 30 ng of iRFP expression plasmid were transfected using 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturer’s protocol. Endogenous gene activation was done similarly but with 200 ng of dCas9–VPR plasmid and 50 ng of sgRNA expression plasmid only.
Hu J.H., Miller S.M., Geurts M.H., Tang W., Chen L., Sun N., Zeina C.M., Gao X., Rees H.A., Lin Z, & Liu D.R. (2018). Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 556(7699), 57-63.
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3

CRISPR Gene Editing in HEK293T and U2OS Cells

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HEK293T cells seeded on 48-well collagen-coated BioCoat plates (Corning) were transfected at approximately 70% confluency. 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (ThermoFisher Scientific) per well according to the manufacturer’s protocol. 500 ng of BE and 250 ng of sgRNA expression plasmids were transfected into U2OS cells using a Lonza 4D-Nucleofector with the DN-100 program according to the manufacturer’s protocols.
Kim Y.B., Komor A.C., Levy J.M., Packer M.S., Zhao K.T, & Liu D.R. (2017). Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nature biotechnology, 35(4), 371-376.
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4

Efficient Genome Editing in Cell Lines

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HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 75% confluency. Briefly, 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturer’s protocol.
HAP1 and HAP1 UNG− cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit S (Lonza) according to the manufacturer’s protocol. Briefly, 4 × 105 cells were nucleofected with 300 ng of BE and 100 ng of sgRNA expression plasmids using the 4D-Nucleofector program DZ-113.
Komor A.C., Zhao K.T., Packer M.S., Gaudelli N.M., Waterbury A.L., Koblan L.W., Kim Y.B., Badran A.H, & Liu D.R. (2017). Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Science Advances, 3(8), eaao4774.
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5

Base Editor Protein Transfection in HEK293T Cells

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HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in 250 μl an antibiotic-free medium and transfected at ∼70% confluency. Base editor protein was incubated with 1.1 × molar excess of the necessary sgRNA at room temperature for 5 min. The complex was then incubated with 1.5 μl Lipofectamine 2000 (Thermo Fisher) and transfected according to the manufacturer's protocol for plasmid delivery. Unless otherwise noted, BE protein was added to a final concentration of 200 nM (based on a total well volume of 275 μl).
Rees H.A., Komor A.C., Yeh W.H., Caetano-Lopes J., Warman M., Edge A.S, & Liu D.R. (2017). Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nature Communications, 8, 15790.
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6

Transfection of HEK293T Cells

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HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in an antibiotic-free medium and transfected at ∼70% confluency. Unless otherwise noted, 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (Thermo Fisher) per well according to the manufacturer's protocol.
Rees H.A., Komor A.C., Yeh W.H., Caetano-Lopes J., Warman M., Edge A.S, & Liu D.R. (2017). Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nature Communications, 8, 15790.
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7

CRISPR-Mediated Gene Editing and Activation in HEK293T Cells

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HEK293T cells were seeded on 48-well poly-D-lysine-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. For genomic DNA cutting or base editing, 750 ng of Cas9 or BE3 and 250 ng of sgRNA expression plasmids were transfected using 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturer’s protocol. For GFP activation, 200 ng of dCas9–VPR plasmid, 50 ng of sgRNA expression plasmid, 60 ng of GFP reporter plasmid, and 30 ng of iRFP expression plasmid were transfected using 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturer’s protocol. Endogenous gene activation was done similarly but with 200 ng of dCas9–VPR plasmid and 50 ng of sgRNA expression plasmid only.
Hu J.H., Miller S.M., Geurts M.H., Tang W., Chen L., Sun N., Zeina C.M., Gao X., Rees H.A., Lin Z, & Liu D.R. (2018). Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 556(7699), 57-63.
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8

CRISPR Gene Editing in HEK293T and U2OS Cells

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HEK293T cells seeded on 48-well collagen-coated BioCoat plates (Corning) were transfected at approximately 70% confluency. 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (ThermoFisher Scientific) per well according to the manufacturer’s protocol. 500 ng of BE and 250 ng of sgRNA expression plasmids were transfected into U2OS cells using a Lonza 4D-Nucleofector with the DN-100 program according to the manufacturer’s protocols.
Kim Y.B., Komor A.C., Levy J.M., Packer M.S., Zhao K.T, & Liu D.R. (2017). Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nature biotechnology, 35(4), 371-376.
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9

CRISPR-Cas9 Gene Editing Protocol

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Sixteen to twenty-four hours before transfection, HEK293T and Neuro-2A cells at more than 90% viability were seeded on 48-well poly-d-lysine-coated plates (BioCoat plates, Corning) at a density of 30,000–40,000 cells per well. Cells were transfected with 1 μl of Lipofectamine 2000 (Thermo Fisher Scientific) and 750 ng of PE plasmid, 250 ng of pegRNA plasmid and 83 ng of sgRNA plasmid. For conditions delivering MLH1dn, 100 ng of additional plasmid was transfected. For 96-well plate (Corning) transfections, 15,000–20,000 cells were plated 16–24 h before transfection, and 200 ng of PE, 40 ng of pegRNA and 13.3 ng of nicking guide with 0.5 μl of Lipofectamine 2000 (Thermo Fisher Scientific) were used. For intein-split PE plasmid transfections, total PE plasmid was reduced to 50 ng. Seventy-two hours after transfection, genomic DNA (gDNA) was isolated with 75–150 μl of lysis buffer (10 mM Tris-HCl pH 8.0, 9.05% SDS, 25 μg ml−1 proteinase K (Thermo Fisher Scientific)) at 37 °C for 1 h, followed by 80 °C for 30 min.
Davis J.R., Banskota S., Levy J.M., Newby G.A., Wang X., Anzalone A.V., Nelson A.T., Chen P.J., Hennes A.D., An M., Roh H., Randolph P.B., Musunuru K, & Liu D.R. (2023). Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nature Biotechnology, 42(2), 253-264.
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10

Base Editor Protein Transfection in HEK293T

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HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in 250 µl of an antibiotic-free medium. After 12 h, HEK293T cells were transfected at ~70% confluency. Base editor protein was incubated with 1.1 times molar excess of the necessary sgRNA at room temperature for 5 min. In parallel, Cas9 protein was incubated with 1.1 times molar excess of sgRNA and different specified molar of ssDNA at room temperature. We observed that higher sgRNA concentration with constant BE3 concentration did not increase base editing efficiency by HTS in vitro. The complex was then incubated with 1.5 µl Lipofectamine 2000 (Thermo Fisher) and transfected according to the manufacturer’s protocol for plasmid delivery. BE3 and Cas9 protein were added to a final concentration of 200 nM (based on a total well volume of 275 µl).
Yeh W.H., Chiang H., Rees H.A., Edge A.S, & Liu D.R. (2018). In vivo base editing of post-mitotic sensory cells. Nature Communications, 9, 2184.
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