In vitro and biochemical assays
The ELISpot assay is a simple and highly sensitive assay for analysis of cell activation at the single-cell level. For IFNγ ELISpot, on day 1, plates are coated with the antibody. On day 2, cells are extracted from organs and plated out, in addition to in vitro stimulants. On day 3, secondary antibody is added, and enzyme conjugate and substrate is developed and the IFNγ is stained. It will be washed and dried overnight, and then it will be read by an ELISpot reader.
Mouse 1 is a control and mouse 2 is treated with compounds. (Figure on right).
Cell survival assay
To test the toxicity of the compounds, we test them using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Here, we show MTT calorimetric assay showing dose-dependent inhibition of lung adenoma cell growth after exposure to cimetidine in vitro. (Figure on left)
[Marina Sirota, J. T.-C. (2011). Discovery and Preclinical Validation of Drug Indications Using Compendia of Public Gene Expression Data.Science Translational Medicine, Vol 3 Issue 96 96ra77.]
Fluorescent Activated Cell Sorter (FACS)
Cells are stained with antibodies with fluorophores with characteristic peak excitation and emission wavelengths. Cells can be stained for both intracellular and surface markers. Cells can also be fixed per user’s request. We can stain, analyze samples and generate reports using FlowJo software.
In Bradford assay, standard Bovine Serum Albumin (BSA) will be prepared as controls, and protein concentration will be calculated using standard curve values.
Myeloid-Derived Suppressor Cell (MDSC) isolation and T cell suppression assay
Myeloid-Derived Suppressor Cells (MDSCs) can be tested to study their effects on T cell proliferation in response to immunotherapy. MDSCs can be collected from various organs including spleen, bone marrow, lymph node and tumor and will be isolated into single cell suspension using enzyme digestion. Then cells will be purified and be stained with, for example, Ly6C, Ly6G, Gr1 to confirm the purity. Effector T cells will be collected from naïve mice, stained with Carboxyfluorescein succinimidyl ester (CFSE), stimulated, and co-incubated with MDSC cells for 3-5 days. After the incubation, FACS assay will be performed for the proliferation analyses based on CFSE intensity.
The FACS histogram on the right shows results of an effector T cell suppression assay. The green lines and black lines represent proliferation of effector T cells incubated with a type of MDSC, as indicated by a cell proliferation marker, CFSE (at 10 μM).
Regulatory T cells (Treg) isolation and T cell suppression assay
Regulatory T cells (Treg) can be tested for their functional roles in T cell proliferation in response to immunotherapy or chemotherapy. Tregs can be collected from different tissues including spleen, bone marrow, lymph node and tumor, and will be isolated into single cell suspension. Then cells will be purified and stained with T cell markers (CD3, CD4 and CD25) to confirm the purity. Effector T cells will be collected from naïve mice, stained with Carboxyfluorescein succinimidyl ester (CFSE), stimulated, and co-incubated with Treg cells for a few days. FACS will be run for further experiment.
We do a serial dilution for standards to create a standard curve. Then we add supernatant from cells or from mice blood and buffer to the wells. Next, wash and read the plate.
Sphere formation assay
Sphere formation assays are used to identify factors that control stem cell growth or their capability of self-renewal. Cells are dissociated into single cell suspension by pipetting and passing through a cell strainer. Single cells are then seeded in serum-free specialty medium specific for tissue types. Sphere should grow in 3-7 days. Afterwards, we subculture every 7 days and start the treatment. After 3-4 times of subculture, we stop passaging because cells may differentiate.