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Set yourself up for success with HAP1 cell lines



Using ready-to-go cell lines, such as Horizon’s HAP1 knockout cells, can save time and money in the lab. They let you skip the design and clonal isolation steps--allowing you to spend less time creating your research tool, and more on your experimental question. If you are new with the culture of HAP1 cells, following these tips will help you to have a successful experience working with them.

Scientist working in cell culture hood

 

1. Keep lines separated
HAP1 knockout (KO) cells are provided as an isogenic pair with the wild-type parental cells as a control for your downstream experiments. To avoid contamination of the KO cell line with the wild type cell line, avoid sharing cell culture medium between the two cell lines. It is strongly recommended that the process of thawing, passaging the cells, and freezing of the aliquots is done in separate protocols. Keeping the WT and KO cells under the hood at the same time could lead to cross-contamination if a pipette or cell culture regent is accidentally shared between the cells.

2. Maintain media controls
Control your reagents for bacterial, fungi, or mycoplasma contamination before culturing your received cells. Bacterial and fungi contamination can be checked by culturing an aliquot of cell-free medium overnight at 37°C for 1 to 3 days. Mycoplasma contamination can be checked by using a designated kit. 

3. Prepare ahead for thawing
To obtain a reasonable revival rate from frozen HAP1 aliquots, it’s best to prepare all the reagents, cell culture bottles, and material before adding the cells in the 37°C water bath. Once the cells are starting to thaw, but there is still a frozen pellet, clean the outside of the cryotube with 70% ethanol, open the cryovial and remove the cells with a 2 ml pipette.  Carefully, then, introduce the pipette into 10 ml of pre-warmed cell culture in a media tube, and slowly release the content of the pipette into the medium. Resuspend the cells carefully and keep them in culture as described in the HAP1 cell culture guidelines.  

4. Validate your assay
HAP1 KO cell lines are verified at the genomic level. For that reason, when you check the KO status of your cell line, please use a monoclonal antibody that has been well characterized to recognize the protein of interest and also binds downstream of the known gene editing event; and test it in your functional assay for your protein of interest.  Examples of functional assays can be protein-protein interaction (1), phenotype (2), affinity chromatography and immunoprecipitation (3), or DNA footprinting (4), among others

5. Maintain ploidy
HAP1 cells can sometimes spontaneously switch to a diploid state. Further, there are fundamental differences between the behavior of haploid and diploid HAP1 cells (5). Therefore, we suggest ploidy is monitored and cell sorting can be performed to maintain haploid status, unless diploidy is preferred based on the assay being performed.  

If you believe you need further assistance with your cell line, please contact us, there is a team of technical support agents that will help you with any additional questions.

Authors: Ivonne Rubio, Ph.D. | Scientific Support Specialist 3 and Travis Hardcastle, MS | Product Manager

 

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References:
1. S Xing, N Wallmeroth., et al. Techniques for the Analysis of Protein-Protein Interactions in Vivo. Plant Physiology 171, 727–758 (2016)
2. M Boutros, F Heigwer., et al. Microscopy-Based High-Content Screening. Cell 163, 1314-1325 (2015)
3. C Tomomori-Sato, S Sato., et al. Immunoaffinity Purification of Protein Complexes from Mammalian Cells. Methods Mol Biol 977, 273–287 (2013)
4. DJ.Galas and A Schmitz. DNAase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acid Research 5, 3157-3170 (1978)
5. T Beigl, I Kjosås, et al. Efficient and crucial quality control of HAP1 cell ploidy status. Biology Open. 9, (2020)