Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).
Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).
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 (GSK3š-i, TGFš-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-š3) 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.
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Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.
An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.
An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.
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