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CRISPR/Cas9 Transfection Optimization

Efficient delivery of the CRISPR/Cas9 protein complex can be achieved efficiently through the use of cationic lipid-based transfection reagents. Cells may undergo electroporation to increase the probability of successful transfection. Once inside a target cell, the CRISPR/Cas9 protein complex requires no additional stimulation to start modifying the cell’s genome. Genome modification will occur within one day of delivery, while gene and protein expression follows in approximately twelve hours. Altogen recommends using high-purity transfection reagents to minimize the chance of introducing undesired DNA into the cell. Proper protocols should be followed to ensure the purity of sgRNA, which guides the Cas9 protein to a particular location in a cell’s genome. Researchers wishing to add genes to DNA should take precautions to ensure that the repair template is introduced along with the CRISPR/Cas9 complex into the cell during transfection. If a plasmid is used to deliver the CRISPR/Cas9 complex into specific cell line of primary cell type, we recommend using one of Altogen Biosystems cell line optimized transfection kits to encapsulate the plasmid and deliver it into a cell.

Plasmid DNA and siRNA transfection protocol optimization

There are several experimental parameters that affect transfection efficiency and associated cell viability for siRNA and plasmid DNA transfection experiments: Transfection protocol (method and conditions) Health of cultured cells Purity and concentration of pDNA and/or siRNA Controls Transfection protocol Optimized transfection protocols are available for over 100 cancer cell lines and primary cell types (see… Continue Reading

Transfection optimization

The most vital aspect of a transfection is ensuring all conditions are optimized, including transfection reagent volume, oligo concentration, cell viability and passage number, lack of negative control activity, activity of positive control and calibrated incubator temperature, humidity and percentage of CO2.  Even if all these parameters are correctly addressed, transfection efficiency must be determined… Continue Reading

Importance of cell passage number and cell confluency for efficient transfection

The viability, confluency and passage number are all vital parameters for a successful transfection.  Here are helpful hints for each of these aspects: Viability Viability is the percentage of living cells in a suspension Determined using trypan blue exclusion Cells are considered to be healthy if viability is greater than 90% Confluency Confluency is the… Continue Reading

Where to purchase cancer cell lines?

Cancer cell lines must be purchased from trustworthy vendors (i.e. ATCC, Sigma-Aldrich, DSMZ), along with signing licenses and agreements.  These large cell bank vendors have established good quality control processes, document controls and facilities that are able to offer well characterized cell lines. Continue Reading

Caspase-3/7 cell line screen for test compound

Caspase 3/7 activity is a cell based assay that measures apoptosis.  Commercially available reagents enable high-throughput screening of apoptosis inducing test compounds or contract research organizations can readily perform the compound screening.  A brief overview of the protocol is as follows: Cells in logarithmic growth are seeded in a 96-well plate Depending on the scope… Continue Reading

Tissue culture using flasks vs dishes for cell lines

Flasks and dishes that are cell culture coated are both acceptable consumables to use for cell culture.  Employing sterile techniques allows an experienced scientist to use either item, with pros and cons listed here for each system. Filtered Flasks Narrow opening translates to a lesser chance of contaminant entering the flask Ideal for long term… Continue Reading

Cell passage: How to correctly dilute and split cultured cells

There are many different cell culture techniques. Utilizing correct cell passaging methods is important to keep cell line in exponential growth curve, therefore making it a good model as a host for transfection experiments. “Cell passage” is a term used by other scientists to demonstrate the following process: Wash cells with PBS Detach cells from… Continue Reading

How to ship cells as a live culture?

Shipping cells to collaborators can be accomplished by either sending them a frozen aliquot on dry ice or by sending a live culture.  Sending a live culture has the advantages of the cells continuing their exponential growth to aid in timing of experiments.  This is accomplished by completely filling the flask with culture medium, closing… Continue Reading

How to determine cell viability?

Cell viability is a calculation of the number of viable or living cells within the total number of cells.  Although there are now commercially available alternatives to the historical method, trypan blue exclusion is the tried and true method commonly used in the lab.  Briefly, trypan blue dye is added to a cell suspension and… Continue Reading

How to determine mycoplasma contamination?

Mycoplasma contamination is a common occurrence and labs must be vigilant in monitoring and combating contamination issues.  Mycoplasma are bacteria extremely difficult to detect in cultured cells by visual inspection.  Many labs do not incorporate recurring mycoplasma testing until the lab experiences a contamination incident, at which time cleaning and testing procedures are put in… Continue Reading

Even cell distribution in the 6-well plate instead of a ring of cells around the edges?

Performing cell culture experiments in round well plates can often lead to troubling spatial dispersion of the cells.  This can be attributed to culture incubators having high vibrations, non-level incubators or bad practices such as swirling the plate after adding cells.  In order to achieve even distribution of cells in the wells of culture plates,… Continue Reading

Is it necessary to synchronize cells?

Exponentially growing cultured cells are growing at difference cell cycle stages, termed asynchronous.  Scientists are vigilant in their techniques to ensure they are using non-confluent cells prior to their experiments; thus, synchronization is not commonly employed.  However, synchronizing your cells can help improve the results of certain cell based assays such as viral integration or… Continue Reading

Low cell viability due to the freezing or thawing of cancer cell lines

Freeze slow, thaw fast.  The process of freezing and thawing cancer cell lines requires extreme attention to protocol.  For freezing, a good freezing media that contains DMSO is added to the correct number of cells following the manufacturer’s instructions.  The vital part of the freezing process is to ensur that the freezing process is slow. … Continue Reading

How much antibiotic required for stable cell selection?

Stable cell lines are a crucial laboratory tool that over-expresses a gene of interest in order to study gene functions, screen experimental drugs or produce therapeutic proteins (i.e. recombinant antibodies).  The cell lines will divide and continue to express the inserted transgene.  Briefly, exogenous plasmid DNA is transfected into a host cell line, which is… Continue Reading

Quantitate in vitro anti-proliferation experiments using a metabolic assay (Alamarblue, MTT) vs measuring protein abundance (Sulforhodamine B)

The need to measure cell proliferation effects is vast, including testing the effects of growth factors, novel pharmacological agents, cytotoxicity assessment or investigating cell activation.  Cell proliferation assays utilize a stain or substrate to make a correlation between the readout and the number of remaining cells post-treatment.  However, the means of assessing cell number varies… Continue Reading

What is the benefit of using an electroporation buffer

The process of electroporation exposes the cells to a high-voltage pulse of electricity to disrupt the phospholipid bilayer of the cell membrane causing the formation of temporary pores.  Any charged molecules (e.g. RNA, DNA) are forced into the cells thru the pores. Electroporation buffers are formulations that mimic cellular cytoplasm composition; thus, enhancing pore resealing… Continue Reading

Difference between technical and biological replicates

The basic definitions of technical and biological replicates are as follows: Technical replicates: a test performed on the same sample multiple times; i.e., if there are triplicate non-treated samples, a technical replicate would be testing sample #1 of the non-treated multiple times Biological replicates: a test performed on biologically distinct samples representing an identical time… Continue Reading

What is the best method to detach cultured cells?

Cultured adherent cells routinely need to be detached and collected for counting or passaging.  Detaching cells can be accomplished by either mechanical or enzymatic methods. Mechanical: cell scraping is a good option for cells that are sensitive to trypsin but can cause damage to cells; also can be used when collecting cellular components for western… Continue Reading

Forward Transfection or Reverse Transfection?

Forward and reverse transfection protocols each have their significant uses in research.  The main protocol difference between forward and reverse transfection is whether or not the cells are plated the day before transfection (as in forward transfection) or seeded at the same time of the transfection.  Forward transfection is commonly used in situations where the… Continue Reading

Stable transfection reagents and techniques

Stable transfection, sometimes called permanent transfection, is the integration of plasmid DNA into the chromosome of cancer cell’s DNA.  The creation of a stable cell line enables the researcher to analyze long term effects of the introduced gene.  Downstream studies incorporating the stable cell line includes overexpression of the gene insert for protein production, cell… Continue Reading

What transfection controls do I need to include in my experiment?

Scientists understand the importance of controls in experiment. There are at least three transfection controls that should be included on every transfection plate: a positive control, negative control and non-treated control. Positive Controls Transfecting a positive control ensures that the system being utilized is working and the delivery conditions are optimal.  The results of the… Continue Reading

What is the best antibiotic to use for stable cell selection in mammalian cells?

There is a long list of antibiotics available to researchers when applying selective pressure in the creation of a stable cell line. The choices include zeocin, hygromycin, blasticidin, puromycin and geneticin (G418). Researchers use different antibiotics due to cost or availability in their lab. However, in reference to using antibiotics for the creation of a… Continue Reading

How do I increase transfection efficiency?

Transfection is considered as a major laboratory method to integrate protein, RNA and DNA molecules into tissues and cells. Delivery of plasmid DNA molecules containing gene inserts, messenger RNA and small interfering RNA molecules that regulate gene expression (i.e. microRNA, siRNA) into the primary cells and cancer cell lines have been extensively utilized by scientists.… Continue Reading