Director: Shivalingappa Swamynathan, PhD
Tissue Culture Services
Basic Cell Culture
- Thawing cells
- Maintaining cells
- Large culture scale-up
- Subculturing for experiments
- Cell counts
- Cell cryopreservation
- Cryostorage bank
- Custom media assembly (SHEM, antibiotic-free media, serum-free media)
Advanced / Specialized Culture Services
- Stem Cells
- Mycoplasma screening of reagents cell lines
- Endotoxin screening of reagents / cell lines
- Organ culture (whole rabbit corneas)
- Wound healing assays (whole corneas, agarose strips, agarose dots)
- Rabbit primary ocular cell culture (endo, epi, stroma, conjunctiva, TM)
- Human primary ocular cell culture (endo, epi, stroma, TM, corneal stromal stem cells)
- Bovine primary ocular cell culture (keratocytes)
- Make stably transfected cell lines (via TurboFect, FuGene, L2K)
- Hybridoma services (culture cells and collect supernatant, concentrate antibody, purify antibody, modify with various tags)
- Recombinant virus isolation – Kip’s team makes the vector, I do the transfection, isolate recombinants, and create a crude primary isolate stock.
- Crude stock amplification
- Three-tier virus cryostorage bank creation
- Virus purification
- Plaque assays – for titers of stocks, eye swabs, TGs, or other brain areas, growth kinetics
Paraffin Histology (FFPE)
- Protocol Formation – determine scientifically how long to fix, process, what wax is best, how long to stain for, appropriate dilutions of antibodies, etc.
- Tissue Fixation
- Tissue Processing
- Paraffin Embedding
- Sectioning on rotary microtome and mounting onto slides
- Chemical Staining (H&E, acetylcholinesterase, blueing)
- Immunohistochemical Staining (DAB)
Frozen Histology (Cryostat)
- Protocol Formation – determine scientifically how long to fix, infiltrate with sucrose, best cutting temperatures, how long to stain for, appropriate dilutions of antibodies, etc.
- Tissue Fixation (sometimes occurs after cutting, sometimes before)
- Sucrose Infiltration
- OCT-cassette Embedding
- Sectioning on Cryostat microtome
- Mounting onto slides
- Chemical Staining (H&E, acetylcholinesterase, blueing, DAPI)
- Immunohistochemical Staining (DAB, fluorescent)
- Chemical Staining – gentian violet, alizarin red, fluorescein, DAPI, trypan blue
- Immunocytochemistry –DAB, fluorescent
- in specific protocols (e.g. how best to grow HEK cells)
- in the principals of certain techniques (e.g. aseptic technique)
- for fundamentals of the scientific method (constructing a well-controlled experiment)
- Lot testing (of FBS, alternative companies from whom to source media)
- Pursue training – I attend seminars and hands-on courses related to projects I do for you.
- Attend your lab meetings to better understand your projects, and to add fresh insights.
- Encourage collaborations between you and other investigators.
- I buy some basic reagents (e.g. trypsin, PBS) for services I perform on your behalf.
- Maintain the equipment I use to perform your projects (e.g. incubators, water baths).
Departmental Resource Oversight
- Dark Room
- Milli-Q Water System
- Cryostat refrigerated microtome
EEINS-928, 203 Lothrop Street, Pittsburgh, PA 15213