96 well optical bottom microplate

Manufactured by Greiner

Sourced in Germany

The 96-well optical-bottom microplate is a laboratory equipment designed for various applications that require optical measurements. It features a transparent bottom that allows light to pass through, enabling optical analysis of samples within the individual wells. This microplate provides a standardized format for containing and analyzing multiple samples simultaneously.

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Lab products found in correlation

Celigo cell imaging cytometer, by Revvity (3 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Nucleocounter nc 200 cell counter, by ChemoMetec (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions)

3 protocols using 96 well optical bottom microplate

Quantifying Cell Viability Using Imaging Cytometry

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Dye master mix was made daily: CD CHO + 8 mM L-glutamine + 5 μg/mL Hoechst-33342 (Life Technologies) + 0.4 μg/mL propidium iodide (Life Technologies). Master mix (200 μL) and cell suspension (3 μL) were mixed in a 96-well optical-bottom microplate (Greiner Bio-One, Frickenhausen, Germany) and cells were incubated for 40 min at RT. Image cytometry analysis was performed on a Celigo Imaging Cell Cytometer (Nexcelom Bioscience, MA, USA). Cells were identified using the blue channel (Hoechst-positive cells), and the red fluorescence channel (propidium iodide) was used to detect dead cells. Total cell density was computed from the total number of identified cells in the well. Cell viability was defined as the percentage of propidium iodide-negative cells. Based on total cell density and viability, viable cell density (VCD) was determined.
For comparison, VCD measurements were performed on a NucleoCounter NC-200 Cell Counter (ChemoMetec, Allerod, Denmark) using Via1-Cassettes™ using a Viability and Cell Count Assay (NucleoView software ver. 1.1.18.1) according to the manufacturer’s instructions.

Hansen H.G., Nilsson C.N., Lund A.M., Kol S., Grav L.M., Lundqvist M., Rockberg J., Lee G.M., Andersen M.R, & Kildegaard H.F. (2015). Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells. Scientific Reports, 5, 18016.

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Characterizing Cas9 Functionality in CHO Cells

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To characterize Cas9 functionality we transfected clonal CHO-S^Cas9 cells with a vector expressing gRNA against Mgat1 and verified indel generation on a pool level by NGS as described previously^{51 (link)} (using gRNA oligo primers MGAT1_gRNA_fwd and MGAT1_gRNA_rev and NGS primers MGAT1_miseq_fwd and MGAT1_miseq_rev listed in Supplementary Table S3). To analyze GFP expression, clonal cells were seeded in wells of a 96-well optical-bottom microplate (Greiner Bio-One) and identified GFP positive cells on the Celigo Cell Imaging Cytometer (Nexcelom Bioscience) using the green fluorescence channel. GFP negative gating was set on the basis of fluorescence emitted from CHO-S wild type cells.

Cour Karottki KJ l.a., Hefzi H., Li S., Pedersen L.E., Spahn P.N., Joshi C., Ruckerbauer D., Hernandez Bort J.A., Thomas A., Lee J.S., Borth N., Lee G.M., Kildegaard H.F, & Lewis N.E. (2021). A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes. Metabolic engineering, 66, 114-122.

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Characterizing CRISPR-Cas9 Gene Editing

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To characterize Cas9 functionality we transfected clonal CHO-S Cas9 cells with a vector expressing gRNA against Mgat1 and verified indel generation on a pool level by NGS as described previously 48 (using gRNA oligo primers MGAT1_gRNA_fwd and MGAT1_gRNA_rev and NGS primers MGAT1_miseq_fwd and MGAT1_miseq_rev listed in Supplementary Table S2). To analyze GFP expression, clonal cells were seeded in wells of a 96-well optical-bottom microplate (Greiner Bio-One) and identified GFP positive cells on the Celigo Cell Imaging Cytometer (Nexcelom Bioscience) using the green fluorescence channel. GFP negative gating was set on the basis of fluorescence emitted from CHO-S wild type cells.

Karottki K.J.l.C., Hefzi H., Li S., Pedersen L.E., Spahn P.N., Joshi C., Ruckerbauer D.E., Bort J.A.H., Thomas A., Lee J.S., Borth N., Lee G.M., Kildegaard H.F., & Lewis N.E. (2020). A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

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