The 293T cells donated by Li Jiang, were grown in 10% fetal bovine serum/Dulbecco's Modified Eagle Medium containing 1% penicillin streptomycin, at 37 °C and 5% CO2. The cell density was adjusted to 5 × 105 using the Automated Cell Counter IM1200 (Countstar, Shanghai, China), and the cells were seeded in culture dishes. When the cell confluency reached 70–80%, we transfected the cells with both WT and mutant plasmids using Lipofectamine 2000 Transfection Reagent (Invitrogen, #11668019) according to the manufacturer's instructions. After 48 h, the transfected cells were harvested for further analysis.
Automated cell counter im1200
Manufactured by Countstar
Sourced in China
The Automated Cell Counter IM1200 is a compact, benchtop device designed for the rapid counting and analysis of cells. It utilizes advanced optical technology to accurately determine cell concentration and viability. The instrument provides users with reliable and reproducible results, making it a valuable tool for various applications in cell biology and life science research.
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Lab products found in correlation
2 protocols using automated cell counter im1200
1
293T Cell Transfection with WT and Mutant Plasmids
2
In vitro Validation of mRNA Splicing
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To investigate the effect of the c.633 + 3 A > G variant on mRNA splicing, an in vitro experimental validation was conducted. The WT plasmids and mutant plasmids were constructed using human genomic DNA as templates. After Sanger sequencing verification, the amplified fragments were reconstituted into pMini-CopGFP (with BamHI and XhoI sites in the vector) using ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). Next, the products were used directly for the transformation of competent TOP10 cells, and positive colonies were selected and amplified. Next, 293T cells density was adjusted to 5 × 105 using the Automated Cell Counter IM1200 (Countstar, Shanghai, China) and transfected by the plasmids following instructions for Lipofectamine 2000 Transfection Reagent (Invitrogen, #11668-019) following the instructions. In the final step, the RNA sample was extracted from cells cultured for 48 h using TRIZOL reagent and subjected to a reverse transcription-polymerase chain reaction (RT-PCR). All the primer-related information and PCR conditions are described in Supplementary Material 1 .
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