Biofluorimetry Lab
Dipartimento di Fisiopatologia Medica
University of Rome 'La Sapienza'
page created and maintained by Leopoldo Silvestroni & Simonetta Palleschi
last update March 3, 2010
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Cell-tissue preparation & Culturing methods and protocols |
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Topics in this page are heavily based on animations and interactivity |
See also our At the Lab bench, Chemistry and Microscopy pages
35S-cysteine Incorporation into cellular protein
3H-thymidine uptake by cultured cells
Axoneme preparation from Chlamydomonas reinhardtii ida 1 mt+
b-Galactosidase Color Assay for Cultured Cells
Bioassay for cytokines by chemotaxis
Bioassays: WHO unit conversion
Bioassays for cytokines with Wnt-3a (cysteine-rich glycoprotein) from brains to bones
Blood cells separation by low-speed centrifugation
Cells:
adhesion evaluation under static conditions
adhesion visualization under well-defined wall shear stress
counting with an hemacytometer (1 - 2 - 3 - 4)
culture media & formulations (1 - 2)
cytotoxicity assay (51Cr method) (neutral red method)
digestion (also tissues) solutions
disruption and subcellular fractionation with nitrogen bomb
dissociation from culture vessels
dissociation from primary tissue
freezing & thawing:
cryopreservation of animal cells
cryopreservation and storage of cell lines
freezer and cell lines personal database (access based)
freezing & thawing cells (1-2-3-4-5-6-7-8-)
growth problems: identifying and correcting
immunofluorescence staining (see the Histocytology & Histocytochemistry section below)
immunoperoxidase staining (see the Histocytology & Histocytochemistry section below)
lysis for extracting nuclear proteins (PDF)
media, trypsin volumes and inoculation density
metabolic labeling (35S-methionine or 32P)
metabolic methylation of protein arginine (PDF)
migration assay (under-agarose method)
proliferation assay with 3H-thymidine
proliferation assay with CyQUANTTM
proliferation assay with MTT (1, PDF) (2, PDF)
synchronization: G1/S Phase Block
synchronization: G2 Phase Block with Hoechst 33342
synchronization: Metaphase Block
synchnronization: G1/S phase double block using thymidine
Brain Membrane Fractions Preparation
Buffers and chemicals in histology
Chambers for live cell examination under a microscope
Chemotaxis (see the Laboratory equipment & techniques page):
spermatozoa, using the Zigmond Chamber
Chick Embryonic Cardiac Myocytes and Fibroblasts culturing
Chick embryonic extrac preparation
Chick Embryonic Myoblasts/Myotubes and Fibroblasts culturing
Conditioned medium for retinal explant culture
Coverslips (se also Slides below):
coating with PDL, lage batch procedure
coating with polyacrylamide substrates
washing with acid and polylysine coating
Culture:
cell mass by measurement of turbidity
establishment of a primary culture
examination of bacterial colonies
prokaryote cell number by dilution plating
transfer of eukaryote suspension cultures
Culture hoods: formaldehyde treatment
Cytoskeleton study with immunoprecipitation
Decontamination of cells from the yeast
Detergent extraction before fixation
Electroporation of single cells
Embedding cells & tissues (see the Histocytology & Histochemistry section below)
Embryonic stem cells:
clones analysis by mini-southern
colonies picking and transferring to 24-well culture dishes
general manual (0.6MB PDF, BRESAGEN Inc, 2004)
in vitro differentiation into:
injection into mouse blastocysts (PDF)
matrigel aliquoting and plating
mouse embryonic fibroblast cells protocols:
fibroblasts preparation harvesting from embryos
metaphase chromosome preparation (PDF)
neurocortical (rat) stem cell protocol
Establishing stable cell lines (specific for GFP-tagged cells, PDF)
Fixation with glutaraldehyde + formaldehyde
G1/S phase block for cell synchronization
G2 phase block with Hoechst 33342 for cell synchronization
G2 Phase Block with Hoechst 33342 for cell synchronization
Growth chamber for time lapse imaging of budding yeast
Human peripheral blood mononuclear cell preparation
Images & movies of Cells in Culture:
F = fluorescence
M = movies
embryonic swiss mouse fibroblast cells (3T3) F
endothelial cells (bovine pulmonary artery line) M
epithelial cells (madin-darby ovine kidney mdok line) M
epithelial cells (normal rabbit kidney RK13 line) M
epithelial cells (rhesus monkey kidney LLC-MK2 line) M
fibroblasts (albino swiss mouse embryo 3T3) M
human bone osteosarcoma cells (U-2 OS) F
human brain glioma cells (U-118 MG) F
human cortical neuronal cells (HCN-1A) F
human fetal lung fibroblast cells (MRC-5) F
human lung carcinoma cells (A-549) F
male rat kangaroo kidney epithelial cells (PtK2) F
mink uterus endometrium epithelial cells (GMMe) F
mouse embryo teratocarcinoma epithelial cells (P19)F
myoblasts (embryonic rat thoracic aorta medial layerA-10 line) M
normal african green monkey kidney epithelial cells (Vero) F
normal african green monkey kidney fibroblast cells (CV-1) F
normal rat kidney epithelial cells (NRK) F
pig kidney epithelial cells (LLC-PK1) F
rat jejunum myenteric plexus enteroglial cells (EGC/PK060399egfr) F
rat kidney mesangial cells (RMC) F
Tahr ovary epithelial cells (HJ1.Ov) F
transformed (Simian Virus 40) african green monkey kidney fibroblast cells (COS-7) F
transformed chicken embryo fibroblast cells (UMNSAH/DF-1) F
transformed mouse cerebellum microglial cells (c8-b4) F
Lysosome Isolation in Isotonic Sucrose
Matrigel invasion assays (1) (2)
Metaphase block for cell synchronization
Metaphase chromosome preparation (1) (2)
Metaphase Chromosome Preparation from Adherent Cells (PDF)
Metaphase Chromosome Preparation from Mouse/Rat Spleen (PDF)
Microdissection of isolated human tissues manual
Microinjection of single cells in culture
Mini-Chamber for regulating gaseous environment during culture
Mitotic chromosomes from mouse peripheral blood
co-culturing neurons and microglia using tissue culture inserts
dissociated cortical neurons culture
dissociated cultures of cerebellar neurons
dissociated superior cervical ganglion neurones
dissociated sympathetic neurons
dorsal horn and dorsal root ganglion neuron co-cultures and microisland c.
dorsal horn neuron monocultures
organotypic hippocampal slice c.
protocol (mike ehlers lab, duke neurobiology)
schwann cells from adult peripheral nerve
Neutrophils and mononuclear cell purification from human blood
Nucleoli Isolation (from HeLa)
Peritoneal macrophage preparation
Permeabilization with Triton X-100
Permeabilization with Triton X-100 + SDS
Platelet isolation from human whole blood
Preparation of nuclear extract from cultured, plated cells
Proliferation assay with HUVECs
Proliferation assay withy TF-1 cells
Purification of human mononuclear cells and neutrophils
Rat liver cell cytosol and/or organelle fractions
RNA preparation from cultures (see the Nucleic acids page)
Sedimentation of cell structures in sucrose (densities of cell structures)
Sedimentation of cell structures in various density gradients
Slides (see also Coverslips above):
coating with poly-L-lysine and silane
Specimen preparation manual (pdf)
Stock solutions and media for tissue culture for Arabidopsis
Stock solutions for tissue culture
Streptolysin O reversible permeabilization of eukaryotic cells
Subculturing monolayer cell cultures (PDF)
Tissue Culture - Bart's Cookbook and Lab Protocols
Trypan blue viability test (1 - 2 - 3)
Western blot standard and for cell lysate protoclos(see the Laboratory equipment & techniques page)