Immunohistochemistry 53BP2 phospho (ser-25) Rabbit IgG Human

Isolating RNA from tissues and paraffin-embedded tissue samples can be challenging due to cross-linking of biomolecules and fragmented nucleic acids. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in presence of RNAse inhibitors. The homogenization process should be carried out on dry ice to maintain the integrity of RNA.

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.

RNA-Seq is a method to sequence RNA by applying Next Generation Sequencing (NGS). The quality of RNA is critical for the success of RNA-Seq. The integrity of RNA is measured by the RNA integrity number (RIN). RIN is computed from RNA electrophoresis and electropherogram profiles (the peak area of the 28S rRNA should be approximately twice the peak area of the 18S rRNA). If you get the RIN value lower than 7, the possibility of getting the low quality of RNA-seq data is high. To get a high quality RNA, it is better to work with fresh samples or snap-freeze the tissues in liquid nitrogen as quickly as possible and store them at -80°C until further use. Make sure designated areas and all your filter tips, microfuge tubes, plastic, and glassware are RNase-free.

Stem cells have the unique ability to self-renew or differentiate themselves into various cell types in response to appropriate signals. These cells are especially important for tissue repair, regeneration, replacement, or in the case of hematopoietic stem cells (HSCs) to differentiate into various myeloid populations. Appropriate signals refer to the growth factor supplements or cytokines that mediate differentiation of various stem cells into the required differentiated form. For instance, HSCs can be differentiated into dendritic cells (with IL-4 and GM-CSF), macrophages (with m-CSF) and MDSCs (with IL-6 and GM-CSF). Human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be first cultured in neural differentiation media (GSK3𝛃-i, TGF𝛃-i, AMPK-i, hLIF) to form neural rosettes, which can be differentiated into neural or glial progenitors (finally differentiated into oligodendrocytes). Neural progenitors can be finally differentiated into glutaminergic (dibytyryl cAMP, ascorbic acid) and dopaminergic (SHH, FGF-8, BDNF, GDNF, TGF-𝛃3) neurons. Thus, it is important to first identify the self-renewing cell line: its source and its final differentiation state, followed by the supplements and cytokines required for the differentiation, and final use. Timelines are another thing that is considered. For instance, it takes 7-10 days to form neural rosettes from iPSCs and 3 days to differentiate neural progenitors to neurons. Finally, the stability for stem cell culture media varies. It is advised to make fresh media every time when differentiating HSCs to myeloid populations, whereas neural differentiation media may remain stable for two weeks when stored in dark between 2-8C.

The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.

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