Wound healing assay can be challenging due to inconsistencies and variations while making a wound on the confluent cell monolayer, consequently leads to wounds of varying sizes and widths. Moreover, this assay causes damage to the cells that are at the edge of the wound, which can prevent cell migration into the wound site and healing. The best solution is to use the standard wound healing assay kits using either combs or inserts to make a defined wound field or gap and prevent the well-to-well variation in these assays.
Wound healing assay can be challenging due to inconsistencies and variations while making a wound on the confluent cell monolayer, consequently leads to wounds of varying sizes and widths. Moreover, this assay causes damage to the cells that are at the edge of the wound, which can prevent cell migration into the wound site and healing. The best solution is to use the standard wound healing assay kits using either combs or inserts to make a defined wound field or gap and prevent the well-to-well variation in these assays.
Wound healing assay can be challenging due to inconsistencies and variations while making a wound on the confluent cell monolayer, consequently leads to wounds of varying sizes and widths. Moreover, this assay causes damage to the cells that are at the edge of the wound, which can prevent cell migration into the wound site and healing. The best solution is to use the standard wound healing assay kits using either combs or inserts to make a defined wound field or gap and prevent the well-to-well variation in these assays.
Wound healing assay can be challenging due to inconsistencies and variations while making a wound on the confluent cell monolayer, consequently leads to wounds of varying sizes and widths. Moreover, this assay causes damage to the cells that are at the edge of the wound, which can prevent cell migration into the wound site and healing. The best solution is to use the standard wound healing assay kits using either combs or inserts to make a defined wound field or gap and prevent the well-to-well variation in these assays.
DNA damage assay is a standard method for determining in-vivo/in-vitro genotoxicity by measuring the breaks in the DNA chain of animal and plant cells. Initial DNA damage leads to cell cycle arrest and, at the final stages, leads to induction of senescence or cell death (apoptosis, necrosis, autophagy, or mitotic catastrophe). Detection of DNA damage from mild to moderate to severe is challenging when studying genotoxicity in the pool of cells. It is favorable to use DNA damage assay kits available for prominent identification of the extent of damage in the analysis.
As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.
As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.
Cell Invasion or Cell Migration assays are technically challenging to set up as the gradient between the two compartments equilibrates in time during the assay. It is also problematic to view cells and for cells to migrate through a non-physiologic polycarbonate or polypropylene filter. Care must be taken while loading the well with cells to form a single cell suspension. Precaution must be taken while trypsinization (under-trypsinization can lead to cell clumping while over-trypsinization could strip off adhesion molecules necessary for migration). This leads to difficulty in getting significant results, when only small numbers of cells cross the filter or when the distribution and/or staining of the cells is uneven.
Microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.
Microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.
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