A hemocytometer, also known as a hemacytometer or counting chamber, is a specialized glass slide with a grid pattern that is used for counting cells, most commonly in the context of cell culture or blood analysis. It is a traditional and widely used tool in cell biology and hematology for quantifying cell concentration, determining cell viability, and assessing cell morphology.

The hemocytometer consists of a thick glass slide with two raised counting areas called “counting chambers” and a coverslip. The counting chambers have an engraved grid pattern, usually composed of large squares that are further divided into smaller squares. The depth of the chamber is standardized, typically at 0.1 mm, allowing the volume of the chamber to be known and used for calculating cell concentration.

To use a hemocytometer for counting cells, follow these steps:

  1. Prepare the cell suspension: Ensure that the cells are well dispersed and that any cell aggregates are broken up to avoid inaccurate counting. If necessary, dilute the cell suspension with an appropriate buffer or medium to achieve a countable concentration.
  2. Load the hemocytometer: Clean the hemocytometer and coverslip with 70% ethanol and allow them to air dry. Place the coverslip on the counting chambers, ensuring that it rests evenly on the raised edges. Carefully pipette a small volume (10-20 µL) of the cell suspension into the chamber by placing the pipette tip at the edge of the coverslip. The capillary action will draw the cell suspension into the chamber. Repeat for the second chamber if desired.
  3. Count the cells: Place the hemocytometer on a microscope stage and focus on the grid pattern using a 10x or 20x objective. Count the cells in a defined number of large squares, usually following a specific pattern (e.g., four corner squares and the central square). Only count the cells that are inside the square or touching the top or right borderlines. Do not count cells touching the bottom or left borderlines to avoid double-counting.
  4. Calculate cell concentration: Use the following formula to calculate the cell concentration:Cell concentration (cells/mL) = (number of cells counted / number of squares) x (dilution factor) x (10^4)

The factor of 10^4 accounts for the volume of the counting chamber (0.1 mm depth and 1 mm x 1 mm square size).

  1. Assess cell viability (optional): If the cell suspension has been stained with a viability dye, such as trypan blue, count the viable (unstained) and non-viable (stained) cells separately and calculate the percentage of viable cells as follows:% viable cells = (number of viable cells / total number of cells) x 100

While hemocytometers are simple and inexpensive tools for counting cells, they do have some limitations, such as potential user variability, the need for manual counting, and the reliance on appropriate cell dilution and dispersion. Alternatives to hemocytometers include automated cell counters and flow cytometry, which can offer higher throughput, greater accuracy, and additional information about cell populations.

Cell Suspension

A cell suspension refers to a liquid mixture containing dispersed individual cells that are not attached to each other or to any surface. In cell culture, a cell suspension can be created by detaching adherent cells from their growth surface, such as a culture flask or plate, and resuspending them in a suitable culture medium. Suspension cultures can also consist of cells that naturally grow in suspension without the need for detachment. Cell suspensions are used in various applications in biological research, biotechnology, and medicine, including cell counting, cell viability assays, flow cytometry, cell sorting, and the production of recombinant proteins or viral vectors.

To create a cell suspension of adherent cells, such as HEK293 cells, the following steps can be performed:

  1. Remove culture medium: Aspirate the culture medium from the flask or plate containing the adherent cells.
  2. Wash the cells: Gently wash the cell monolayer with phosphate-buffered saline (PBS) or another suitable buffer to remove residual culture medium and any dead or detached cells.
  3. Detach the cells: Add an appropriate volume of cell detachment solution, such as trypsin-EDTA, to the flask or plate. Incubate at 37°C for a few minutes to allow the enzymatic detachment of the cells from the surface. Monitor the detachment under a microscope to avoid over-trypsinization.
  4. Neutralize trypsin: Add an equal volume of complete culture medium containing serum to the flask or plate to neutralize the trypsin-EDTA solution. Serum contains trypsin inhibitors that will halt the enzymatic activity of trypsin.
  5. Collect the cells: Gently pipette the cell suspension up and down to ensure that the cells are well dispersed and detached from each other. Transfer the cell suspension to a sterile tube.
  6. Optionally, centrifuge the cells: If necessary, centrifuge the cell suspension at 200-300 x g for 5 minutes to pellet the cells. Aspirate the supernatant and resuspend the cell pellet in fresh culture medium or buffer, depending on the intended application.

For cells that naturally grow in suspension, such as certain lymphocyte or hybridoma cell lines, no detachment step is required. The cells can be directly collected from the culture medium and used for various applications as needed.

HEK293 Cryopreservation

Cryopreservation is the process of preserving cells by freezing them at ultra-low temperatures, typically -80°C or in liquid nitrogen (-196°C). This process allows researchers to store and maintain cell lines, such as HEK293, for long periods, ensuring the availability of viable cells for future experiments and reducing the need for continuous passaging. Cryopreservation is especially useful for maintaining early passages of cell lines, which can help minimize the effects of genetic drift and phenotypic changes that may occur over time.

Here is a general protocol for cryopreserving HEK293 cells:

  1. Harvest the cells: When the HEK293 cells reach around 80-90% confluency, harvest them by detaching the cells from the culture flask using trypsin-EDTA. After incubation, neutralize the trypsin by adding complete growth medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin).
  2. Centrifuge the cells: Transfer the cell suspension to a sterile centrifuge tube and centrifuge the cells at approximately 200-300 x g for 5 minutes to pellet the cells.
  3. Prepare freezing medium: Prepare a cryoprotective medium containing 90% complete growth medium (without antibiotics) and 10% dimethyl sulfoxide (DMSO). DMSO is a commonly used cryoprotectant that helps prevent the formation of ice crystals within cells during the freezing process.
  4. Resuspend the cells: After centrifugation, aspirate the supernatant and gently resuspend the cell pellet in the prepared freezing medium. The cell concentration should typically be between 1-5 million cells/mL, depending on the desired cell density upon thawing.
  5. Aliquot and freeze the cells: Transfer 1-2 mL of the cell suspension to cryovials, label the vials with relevant information (e.g., cell line, passage number, date), and immediately place the vials in a freezing container. The freezing container should provide a controlled cooling rate of approximately -1°C per minute, which can be achieved using a commercially available freezing container or a homemade device, such as a foam container filled with isopropanol.
  6. Store the cells: After a minimum of 4 hours in the -80°C freezer, transfer the cryovials to liquid nitrogen for long-term storage. It is recommended to store the cells in the vapor phase rather than directly in liquid nitrogen to prevent potential contamination.

When needed, HEK293 cells can be thawed and cultured according to standard protocols. It is important to monitor the viability and characteristics of the cells after thawing to ensure consistency and reproducibility in experimental results.

HEK293 Cell Viability

Cell viability refers to the proportion of living cells in a population, and it is an important parameter to assess the health and quality of cultured cells, such as HEK293 cells. Monitoring cell viability is crucial for optimizing cell culture conditions, assessing the effects of experimental treatments, and ensuring the reproducibility of experiments. There are several methods to determine cell viability, and some of the most common methods include:

  1. Trypan Blue Exclusion: This method relies on the principle that live cells have intact cell membranes that exclude the dye trypan blue, while dead cells with compromised membranes will take up the dye. After trypsinizing and resuspending the HEK293 cells, mix an equal volume of the cell suspension with 0.4% trypan blue solution. Load the mixture into a hemocytometer and count the number of unstained (viable) and stained (non-viable) cells under a microscope. Calculate the percentage of viable cells as follows:% viable cells = (number of viable cells / total number of cells) x 100
  2. MTT Assay: The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a colorimetric method based on the reduction of the MTT dye by viable cells, producing a colored formazan product. The intensity of the color is proportional to the number of viable cells. Seed HEK293 cells in a 96-well plate and incubate with MTT reagent according to the manufacturer’s instructions. Measure the absorbance of the formazan product using a microplate reader, and use the absorbance values to calculate the percentage of viable cells.
  3. Flow Cytometry with Fluorescent Dyes: Flow cytometry can be used to assess cell viability by staining HEK293 cells with fluorescent dyes that differentiate between live and dead cells based on membrane integrity or enzymatic activity. Common dyes for assessing cell viability include propidium iodide (PI), 7-aminoactinomycin D (7-AAD), and annexin V. Stain the cells according to the manufacturer’s instructions and analyze the stained cells using a flow cytometer. The flow cytometry data can be used to calculate the percentage of viable cells in the population.
  4. Automated Cell Counters: Some automated cell counters are equipped with cell viability assessment capabilities, which can save time and reduce variability compared to manual methods. These instruments typically use impedance-based measurements or fluorescent dyes to differentiate between live and dead cells. Follow the manufacturer’s instructions for sample preparation and instrument settings to assess the viability of HEK293 cells.

Each of these methods has its advantages and limitations, and the choice of method will depend on factors such as the required accuracy, available equipment, and experimental context. It is important to regularly monitor cell viability during culture and experimental treatments to ensure the quality and consistency of HEK293 cells and the reproducibility of experimental results.

HEK293 Cytogenetics

HEK293 cells, or human embryonic kidney 293 cells, are a widely used immortalized cell line in cell and molecular biology research. The HEK293 cell line was derived in the early 1970s by transfecting human embryonic kidney cells with sheared adenovirus 5 DNA. The resulting cells contained a portion of the adenovirus genome, which provided the cells with their immortalization properties.

Cytogenetics is the study of chromosomes, their structure, function, and behavior in relation to gene inheritance, organization, and expression. In the context of HEK293 cells, understanding their cytogenetics is essential for determining the stability and characteristics of the cell line, which can impact experimental outcomes and the production of recombinant proteins or viral vectors.

HEK293 cells are known to exhibit an abnormal karyotype, which means they have an abnormal number and/or structure of chromosomes. The normal human karyotype consists of 46 chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes (XX in females, XY in males). However, HEK293 cells typically show a hyperdiploid karyotype, with a chromosome count ranging from 60 to 70, and have been reported to contain both human and adenoviral DNA sequences.

Due to the chromosomal instability of HEK293 cells, the karyotype can change over time and with continuous passaging. This instability can potentially affect gene expression, cell behavior, and the reproducibility of experiments. It is crucial to monitor the characteristics of the cell line and passage number to ensure consistency in experimental results.

Despite their abnormal karyotype, HEK293 cells remain a popular choice for various research applications, including gene expression studies, protein production, and viral vector production, due to their high transfection efficiency, rapid growth, and ease of maintenance in culture.

Sphingosine-1-phosphate Receptors EDG1

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator involved in various physiological processes, such as cell proliferation, survival, migration, and angiogenesis. S1P exerts its effects by binding to a family of G protein-coupled receptors (GPCRs) known as sphingosine-1-phosphate receptors (S1PRs). There are five known subtypes of S1PRs, designated S1P1 to S1P5. EDG1 is an older name for the S1P1 receptor, also known as S1PR1.

S1PR1 is widely expressed in different tissues and cell types, with particularly high expression in the vascular endothelium, immune cells, and the central nervous system. The activation of S1PR1 by S1P initiates various intracellular signaling pathways, depending on the specific cell type and context. Some of the physiological processes regulated by S1PR1 include:

  1. Vascular development and function: S1PR1 plays a critical role in the development and maintenance of the vascular system. It is involved in endothelial cell migration, proliferation, and barrier function, which are essential for blood vessel formation and integrity.
  2. Immune cell trafficking: S1PR1 is expressed in various immune cells, such as T cells, B cells, and dendritic cells, and is involved in the regulation of their trafficking and migration. The gradient of S1P in tissues and blood modulates the movement of immune cells between lymphoid organs and peripheral tissues, which is essential for immune surveillance and response.
  3. Central nervous system (CNS) function: S1PR1 is expressed in various CNS cell types, such as neurons, astrocytes, and oligodendrocytes, and is involved in the regulation of neuronal survival, migration, and synaptic plasticity. S1PR1 signaling has also been implicated in neuroinflammation and neurodegenerative diseases.

Due to its involvement in various physiological processes and its association with several pathological conditions, such as autoimmune diseases, inflammation, and cancer, S1PR1 has been considered a potential therapeutic target. Fingolimod (Gilenya), an FDA-approved drug for the treatment of multiple sclerosis, is a modulator of S1PRs, including S1PR1. Fingolimod acts as a functional antagonist of S1PR1, causing the internalization and degradation of the receptor, which reduces the migration of immune cells into the CNS and dampens the autoimmune response.

The development of more selective modulators of S1PR1 and other S1PRs may offer new therapeutic opportunities for the treatment of various diseases involving S1P signaling, such as inflammation, cancer, and vascular disorders. However, further research is needed to better understand the molecular mechanisms underlying S1PR1 activation, function, and regulation, which may facilitate the development of selective and effective therapeutic interventions targeting S1PR1 and other S1P receptors.

Transient Receptor Potential TRPC1

Transient receptor potential (TRP) channels are a family of non-selective cation channels that play crucial roles in various physiological processes, such as sensory perception, cell signaling, and ion homeostasis. TRP channels can be classified into several subfamilies, one of which is the canonical TRP subfamily (TRPC). TRPC1 is a member of the TRPC subfamily and is widely expressed in different tissues, including the heart, brain, kidneys, and smooth muscle.

TRPC1 is a plasma membrane protein that forms a non-selective cation channel permeable to both monovalent and divalent cations, such as sodium (Na+), potassium (K+), and calcium (Ca2+). The activation of TRPC1 can be triggered by various stimuli, such as receptor-mediated signaling, changes in membrane potential, or alterations in intracellular Ca2+ levels. The exact mechanisms of TRPC1 activation are still not fully understood and are thought to involve interactions with other proteins, lipids, or TRP channel subunits.

TRPC1 has been implicated in several physiological processes, including:

  1. Calcium signaling: TRPC1 is involved in the regulation of intracellular Ca2+ levels, which is essential for various cellular functions, such as muscle contraction, neurotransmitter release, and gene expression. Dysregulation of TRPC1-mediated Ca2+ signaling has been associated with various pathological conditions, including cardiac hypertrophy, kidney disease, and neurodegeneration.
  2. Neuronal development and function: TRPC1 is expressed in neurons and has been implicated in neuronal growth, differentiation, and synaptic plasticity, which are critical for learning and memory.
  3. Smooth muscle contraction: TRPC1 is involved in the regulation of smooth muscle tone and has been implicated in the control of vascular and airway smooth muscle contraction, which is relevant to blood pressure regulation and respiratory function.
  4. Cell proliferation and migration: TRPC1 has been implicated in the regulation of cell proliferation and migration, which are important processes for tissue repair, regeneration, and tumorigenesis.

Due to its involvement in various physiological processes and its association with several pathological conditions, TRPC1 has been considered a potential therapeutic target. However, the development of selective modulators of TRPC1 and other TRP channels has been challenging, as these channels often share similar structures and functions. Further research is needed to better understand the molecular mechanisms underlying TRPC1 activation, function, and regulation, which may facilitate the development of selective and effective therapeutic interventions targeting TRPC1 and other TRP channels.

Corticotrophin Releasing Factor

Corticotropin-releasing factor (CRF), also known as corticotropin-releasing hormone (CRH), is a peptide hormone and neurotransmitter produced primarily in the hypothalamus, a region of the brain involved in the regulation of various physiological processes, including stress responses, reproduction, and energy balance.

CRF plays a crucial role in the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which is the body’s primary stress response system. When the brain perceives a stressor, CRF is released from the hypothalamus into the portal blood system, which connects the hypothalamus with the anterior pituitary gland. Upon reaching the anterior pituitary, CRF stimulates the release of adrenocorticotropic hormone (ACTH) into the bloodstream. ACTH then travels to the adrenal glands, located on top of the kidneys, and stimulates the production and release of cortisol, a glucocorticoid hormone that helps the body cope with stress by increasing blood sugar levels, suppressing the immune system, and modulating metabolism.

Apart from its role in the HPA axis, CRF also functions as a neurotransmitter in various brain regions, where it is involved in the regulation of mood, anxiety, appetite, and energy homeostasis. Dysregulation of CRF signaling has been implicated in several psychiatric and neurological disorders, including depression, anxiety, eating disorders, and addiction. As a result, CRF and its receptors have been considered as potential therapeutic targets for the development of drugs to treat these conditions.

There are two main types of CRF receptors, CRF1 and CRF2, which are both G protein-coupled receptors (GPCRs) and are expressed in different brain regions and peripheral tissues. While the activation of CRF1 receptors is mainly associated with the stress response and the development of anxiety- and depression-like behaviors, the activation of CRF2 receptors has been linked to the regulation of feeding, social behavior, and stress recovery. Therefore, the development of selective CRF receptor agonists or antagonists may hold promise for the treatment of stress-related disorders and other conditions involving CRF signaling.

Muscarinic Acetylcholine Receptor M3

The muscarinic acetylcholine receptor M3 (M3 receptor) is a subtype of the muscarinic acetylcholine receptors, a family of G protein-coupled receptors (GPCRs) that mediate the effects of the neurotransmitter acetylcholine. These receptors are involved in a wide range of physiological functions in the central and peripheral nervous systems, as well as in non-neuronal tissues.

The M3 receptor is predominantly expressed in smooth muscle cells, exocrine glands, and the central nervous system. It plays a crucial role in various physiological processes, such as:

  1. Smooth muscle contraction: Activation of the M3 receptor in smooth muscle cells, particularly in the airways, gastrointestinal tract, and urinary bladder, leads to muscle contraction. This is responsible for bronchoconstriction, gastrointestinal motility, and bladder emptying.
  2. Exocrine gland secretion: The M3 receptor is involved in the regulation of secretions from exocrine glands, such as salivary, lacrimal, and sweat glands.
  3. Pupil constriction: Activation of the M3 receptor in the ciliary muscle of the eye results in pupil constriction (miosis) and changes in the shape of the lens, which are important for focusing on near objects (accommodation).
  4. Central nervous system functions: The M3 receptor is expressed in various regions of the brain and is implicated in the regulation of cognitive functions, learning, memory, and other neural processes.

Pharmacologically, the M3 receptor can be targeted by both agonists and antagonists. Agonists, such as carbachol and bethanechol, mimic the effects of acetylcholine and can be used to treat conditions like xerostomia (dry mouth) or urinary retention. Antagonists, also known as muscarinic blockers or antimuscarinics, such as tiotropium and darifenacin, block the effects of acetylcholine and are used to treat conditions like chronic obstructive pulmonary disease (COPD), asthma, overactive bladder, and irritable bowel syndrome.

It is important to note that targeting the M3 receptor can cause side effects due to its widespread distribution and involvement in various physiological processes. For example, antimuscarinic drugs can cause dry mouth, constipation, blurred vision, and urinary retention. Therefore, the development of more selective drugs that target specific receptor subtypes or tissues can help minimize side effects and improve therapeutic outcomes.

Serum-Free Culture

Serum-free culture refers to the growth of cells in a culture medium that does not contain any serum, which is the liquid fraction of blood obtained after coagulation and removal of blood cells. Serum, such as fetal bovine serum (FBS), is often used as a supplement in cell culture media to provide essential nutrients, growth factors, and hormones required for cell growth and maintenance. However, the use of serum in cell culture has several drawbacks, including batch-to-batch variability, the risk of contamination, and ethical concerns related to animal welfare.

Serum-free culture offers several advantages over serum-supplemented culture:

  1. Consistency: Serum-free media formulations have fewer batch-to-batch variations, which can lead to more consistent experimental results and improved reproducibility.
  2. Reduced contamination risk: The absence of serum lowers the risk of contamination with pathogens or adventitious agents, such as viruses, mycoplasma, or prions.
  3. Cost-effectiveness: Although serum-free media formulations can be more expensive upfront, they eliminate the need for serum, which can be costly and subject to price fluctuations.
  4. Enhanced protein production: Serum-free culture can result in higher yields of recombinant proteins, as it eliminates serum-derived proteins that may interfere with downstream purification processes.
  5. Ethical considerations: Serum-free culture reduces the reliance on animal-derived products, addressing concerns related to animal welfare and the use of animals in research.
  6. Regulatory compliance: Serum-free culture can simplify regulatory compliance, as it reduces the potential for contaminants and allergens in biopharmaceutical production.

Serum-free media are specifically formulated to provide all the essential nutrients, growth factors, and hormones required for cell growth and maintenance. This can be achieved using a combination of purified proteins, peptides, amino acids, vitamins, and other supplements. Some serum-free media are designed for specific cell types or applications, such as stem cell culture, hybridoma culture, or biopharmaceutical production.

It is important to note that adapting cells to serum-free culture may require a gradual transition and optimization of culture conditions, as cells previously grown in serum-supplemented media may initially experience reduced growth rates or viability. This adaptation process can take several passages or weeks to complete, depending on the cell type and specific serum-free media formulation used.