Consultation on design of antigens and screening strategies
The DFCI Confocal and Light Microscopy Core Facility has several types of microscopy equipment equipment available for use. We have a widefield (non-confocal) inverted microscope that has both transmitted light and epi-fluorescence illumination which can be used for both fixed cell fluorescence imaging or live-cell imaging. In addition, a spinning disk confocal microscope on an inverted Nikon microscope is available. A Leica SP5 laser scanning confocal on an inverted microscope has conventional scanner, a fast resonant scanner, and spectral excitation and emission. Our newest investment is the Zeiss 710 laser scanning confocal / multi-photon / FLIM system.
Spinning Disk Confocal Assisted Use
$115.00/hr (DFCI internal)
$185.00 (external academic)
Leica SP5X confocal Assisted Use
$130.00/hr (DFCI internal)
$200.00 (external academic)
Zeiss 710 with 2-photon laser or FLIM Assisted Use
$165.00/hr (DFCI internal)
$180.00 (external academic)
Enzyme-linked immunosorbent assay (ELISA)
Additional costs for antibodies, ELISA kits, lysates.
Fluorescence Activated Cell Sorting
There has been a recent surge in research involving “humanized” mice, which both underscores the growing need for and the finding that these human developmental and disease models can provide reliable and validated in vivo systems to study complex biological processes. We are developing humanized mouse models for different areas of investigations including regenerative medicine, cancer immunotherapy and stem cell biology, B-cell developmental biology as well as infectious diseases and related vaccine development. For more information please visit our website at www.humouse.org.
Human B-cell Ontogeny and Vaccine Development
We are constructing different types of “humanized mice” for investigations of B-cell ontogeny and their utility in evaluating infectious diseases and cancer vaccines. In one such model, the “BLT” mouse develop a functional human lymphoid system after human stem cell engraftment. We have shown that a single intramuscular immunization with recombinant viral envelope antigens, e.g., HIVgp140 and West Nile Virus envelope (WNV-E) proteins, together with the immune-stimulatory TLR9 agonist results in seroconversion and secretion of antigen-specific human antibodies. In vitro studies with human T-cells recovered from these mice demonstrate suboptimal proliferative responses and loss of co-stimulatory surface proteins ex vivo that could be partially reversed with human interleukin (IL)-2 and IL-7. We are currently investigating whether CD4 helper T-cell functions which are required for optimal B cell maturation can modulated in vivo by the exogenous delivery of human cytokines/growth factors.
In a second study, we performed on the analysis of single-sorted human B cells from humanized NOD/SCID gc-/- (hNSG) mice at both genetic and functional levels. B cells from different stages of development, from early immature in the bone marrow to naïve mature in the periphery were sorted as single cells, the antibody genes cloned and expressed as single chain antibody fragments (scFvFcs). Sequence analysis of the antibody genes suggested an impairment in the checkpoint control mechanisms based on longer HCDR3 regions with high levels of positive charges found in the peripheral naïve mature B cells, a characteristic of auto/polyreactive antibodies. This observation was functionally confirmed by performing autoantigen binding analysis with the purified scFvFcs. Moreover, our findings of poly/auto reactive antibodies were extended to show that many of these antibodies can also bind with high affinity binding to HIV envelope protein. This suggests that the hNSG model (and BLT/GTL models) can be used to study the ontogeny of anti-HIV antibodies at the molecular level.
ChIP-Seq Library Prep – Reagents $230 per sample
ChIP-Seq Library Prep – Labor $960 per 10 samples
Required input: 10ng DNA minimum
RNA-Seq Poly-A Prep – Reagents $70 per sample
RNA-Seq Poly-A Prep – Labor $1,080 per 10 samples
Required input: 100ng total RNA minimum
RNA-Seq DSN Prep – Reagents $135 per sample
DNA - Fragment Library Prep – Labor $1,200 per 10 samples
Required input: 100ng total RNA minimum
DNA - Fragment Library Prep – Reagents $55 per sample
DNA - Fragment Library Prep – Labor $960 per 10 samples
Required input: 1ug DNA minimum
DNA - Mate-Pair Prep – Reagents $380 per sample
DNA - Fragment Library Prep – Labor $1,920 per 10 samples Required input: 10ug DNA minimum
Illumina next generation sequencing
Single end 50 cycle $1,200
Single end 100 cycle $1,600
Paired-end 50 cycle $2,000
Paired-end 100 cycle $2,700
Additional Sample QC Cost per additional sample/library - $50
Monoclonal antibody production
We offer a analytical services for genomic and other ‘omic data including: gene expression, copy number variation, ChIP-chip/ChIP-seq, SNP, and next generation DNA sequencing data.
Some examples of the type of questions we can work with investigators to answer are as follows:
Identify novel gene signatures, and validate them using our large in-house cancer gene expression profile database.
Discover which biological pathways are differentially expressed.
Identify pattern of chromosomal abnormalities and loss of heterogeneity in aCGH data from SNP arrays.
Discover promoters or regulators of gene expression.
Build network models of gene expression, protein-protein interaction and literature data to uncover new biological connections.
Integration of high-dimensional clinical and demographic information with ‘omic data.
Multi-platform analysis e.g. Affymetrix, Illumina, and DASL data.
The Director is available to discuss your project and imaging needs, help troubleshoot sample preparation, optimize the imaging equipment, or assist with equipment in your own lab.
$90.00/hr (DFCI internal)
$155.00 (external academic)
Help with microscopy equipment that you have in your lab. Services available in your own lab include:
a) Mercury bulb replacement: Mercury bulbs need to be replaced after about 200 hours of use for optimal fluorescence microscopy and to ensure safety. Alignment of the bulb also needs to be checked periodically in order to make sure illumination is even across the field of view. I will replace your bulb and show you proper alignment technique. $45 (not including replacement bulb).
b) Halide/mercury fluorescent bulb replacement: The new fluorescent light sources use bulbs that last longer than the older mercury bulbs and are designed to have even illumination with minimal alignment. Bulbs are rated to last 2000 hrs., but many last for less time. The fluorescent light is delivered via what is called a liquid light guide, which decays over time and also requires periodic replacement. $45 (not including replacement bulb or replacement liquid light guide).
c) Halogen/brightfield bulb replacement: The brightfield / white light halogen bulb on your microscope will periodically burn out. The lifetime of these bulbs is variable and much less critical to monitor than mercury bulbs. These are like typical light bulbs that can just be replaced when they burnout. Alignment should be performed each time a user sits down at the microscope (see Koehler Illumination) $45 (not including replacement bulb).
Koehler illumination tutorial
Microscope misalignment can cause many problems with transmitted light (brightfield, phase, Nomarski DIC) and fluorescence imaging. It is essential that each microscopy user understand and be able to perform Koehler alignment, particularly with transmitted light microscopy since it requires realignment of the light source each time the sample or objective is changed. I will show you how to do this alignment on your microscope. $90 per hour.
Objective lens cleaning
Objective lenses must be cleaned on a regular basis to ensure optimal performance. Even with the best microscope and camera, you cannot capture a good image with a dirty lens! I will carefully clean the objectives on the microscope in your lab. $45 for up to five objectives.
Microscopes require regular maintenance to keep them in good working order. Without proper care, poor image quality and damaged equipment could result. This service includes cleaning the condenser, removing the oil and dust from all of the optics, and checking the performance of instrument. $90 per hour. Email me your microscope model for an estimate.
Diagnose a problem with your microscope
If you are having problems with a microscope in your lab, I may be able to help detect a problem. We can determine whether it’s a quick fix or something that may require contacting the appropriate company. Perhaps your fluorescence image is not what you’d expect? or maybe you are having a problem capturing an image like what you can see through the eye pieces? Please contact me. Help that requires extensive time will cost $90 per hour.
Next generation sequencing data analysis
Process Development describes the conversation we have with our clients to customize the steps that will be used in their specific project. This includes antigen design and production, animal species/strain selection and immunization protocol determination, assay selection and/or design, antibody characterization, hybridoma method selection and screening design, or something required that is unique.