MCB Laboratory Research Protocols
Special Notes on Procedures
Waste Disposal Procedures
Special Precautions Needed
Cell Culture Protocols
MCBLP-001CC: Culturing of Human Mesenchymal Stem Cells/hMSCs
This is to culture and start the initial seeding of Human Mesenchymal Stem Cells (hMSCs), which are adherent fibroblast -like cells isolated from bone marrow, adipose, and other tissues.
MCBLP-002CC: Subculturing and expansion of Human Mesenchymal Stem Cells/hMSCs
This is to subculture and passage adherent cell lines in culture. Subculturing, or splitting the cells, produces new cultures with lower cell density than the original culture. By removing the medium and transferring the cells into fresh growth medium, the cells are given fresh nutrients and toxic metabolites are removed, allowing long-term maintenance of the culture.
MCBLP-003CC: Cell Counting with a Hemocytometer
This is to determine the concentration of cells in a given sample or obtain a viable cell count from a suspension which requires the use of a counting chamber called a hemocytometer.
MCBLP-004CC: Thawing Cryopreserved Cells
This is to recover and thaw frozen cells from cryogenic storage and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly.
MCBLP-005CC: Cryopreservation of Cells
This is to properly freeze/cryopreserve cell lines as stocks to structurally preserve intact cells and tissues for long term storage in liquid nitrogen or -150°C freezers. Cryopreserving cultured cells is done in the presence of a cryoprotectant which reduces the freezing point of the medium and also allows a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death.
MCBLP-006CC: Generation of Growth Curve (for Cell Lines)
This is to evaluate the growth characteristics of a particular cell line. From a growth curve, the lag time, population doubling time, and saturation density of the cell culture can be determined.
MCBLP-007CC: Isolation and Culture of Human Gingival Fibroblasts (HGnF)
This is to establish a simple method of culturing viable human gingival fibroblasts from gingival tissue samples.
MCBLP-008CC: Alkaline phosphatase (ALP) Staining
This is to detect and differentiate stem cells based on level elevation of alkaline phosphatase activities on their cell membrane and to test pluripotency status.
MCBLP-009CC: Modified Alkaline Phosphatase (ALP) Quantification Assay
This is to measure ALP activity in biological samples using a simple, direct and HTS-ready colorimetric assay such as BioVision’s Alkaline Phosphatase Assay Kit. ALP catalyzes the hydrolysis of phosphate esters in alkaline buffer and produces an organic radical and inorganic phosphate. Changes in alkaline phosphatase level and activity are associated with various disease states in the liver and bone.
MCBLP-010CC: Nile Red Quantification for Lipids
This is to differentiate hMSCs using a vital staining method such as Nile Red which is used to localize and quantitate lipids, particularly neutral lipid droplets within cells. Using Nile Red staining, differentiation is indicated by red staining of the cytoplasm due to the synthesis of fatty acids involved in milk production.
MCBLP-011CC: Oil Red O Staining
This is to provide a staining method using a fat-soluble diazo dye such as Oil Red O that is commonly used to identify exogenous or endogenous lipid and fat deposits in cells and tissues. This also allows selective staining and detection of neutral lipids within cultured cells.
Gene & Protein Purification, Expression and Analysis Protocols
MCBLP-001GP: Agarose Gel Electrophoresis for Separation of DNA samples
The purpose of agarose electrophoresis is to separate DNA based on size, as well as to visualize it after amplification or DNA extraction (e.g. such as plasmid or genomic DNA extraction). DNA can then be extracted from the gel for further analysis and processing.
MCBLP-002GP: PCR using Platinum® PCR SuperMix by Invitrogen
The purpose is to amplify DNA so that it can be visible for analysis or further processing.
MCBLP-003GP: PureLink® Genomic DNA Extraction by Invitrogen™
The purpose is to extract and purify genomic DNA using the PureLink® Genomic DNA kits by Invitrogen™.
MCBLP-004GP: SDS-PAGE Gel Preparation (for Proteins)
The purpose is to prepare a discontinuous polyacrylamide gel used as a support medium for separating proteins by electrophoresis. A prepared gel of a given acrylamide concentration or type separates proteins effectively within a characteristic range.
MCBLP-005GP: SDS-PAGE for Protein Separation
This is to separate proteins or polypeptides according to their sizes or molecular weights using an electrophoretic method. The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate (SDS) which is used to linearize proteins and to negatively charge the proteins. As a result, negatively charged proteins will migrate towards the positive electrode and will be fractionated by approximate size during electrophoresis.
MCBLP-006GP: SDS-PAGE Staining and Destaining
This is to retain or fix the proteins within the gel matrix and to make the protein bands visible for analysis. Once the gel run is complete, a separate dye such as bromophenol blue will bind to the protein in the polyacrylamide gel for the proteins to be visible during analysis. Excess dye is removed during the destaining step with a wash solution consisting of the fixative solution minus the dye through a diffusion process.
MCBLP-007GP: WESTERN BLOTTING Gel Blot Transfer to Membrane (for Proteins)
This is to transfer proteins from the gel to the membrane support typically nitrocellulose or PVDF. The balance of SDS and methanol in the transfer buffer, protein size and gel percentage will affect transfer efficiency.
MCBLP-008GP: Real-Time PCR (qPCR)
This is to identify genetic activity of specific genes within a group of cells as a response to various influences either from internal or external effects.
MCBLP-009GP: Nucleic acid quantification using spectrophotometric method
This is to measure the concentration of nucleic acid samples using the Eppendorf spectrophotometer with 1 mm cuvette.
MCBLP-010GP: Protein quantification using spectrophotometric method
This is to measure the concentration of protein samples using the Eppendorf spectrophotometer with 1 mm cuvette.
MCBLP-011GP: Polyacrylamide Gel Preparation (for Nucleic acids)
This is to prepare polyacrylamide gels which are used to separate shorter nucleic acids (double stranded DNA of up to 1000 base pairs), based on the concentration used. Different concentrations of acrylamide are necessary to optimize resolution of nucleic acids with different lengths. Gels provide a simple, low-cost way to separate nucleic acids based on size for quantification and purification.
MCBLP-012GP: Polyacrylamide Gel Electrophoresis (PAGE) for Nucleic Acids
This is to pour and run a neutral polyacrylamide gel for nucleic acid samples. Gel electrophoresis is used to separate macromolecules such as DNA, RNA and proteins. DNA fragments are separated according to their size.
MCBLP-013GP: Gel Staining (PAGE for Nucleic Acids )
This is for the visualization of the DNA/RNa bands within the gel corresponding to base pairs with different molecular weights. The most common dye used to make DNA or RNA bands visible for gel electrophoresis is Ethidium bromide (EtBr) which fluoresces under UV light when intercalated into the major groove of DNA (or RNA).
Immunohistochemical Studies Protocols
MCBLP-001IH: Tissue Preparation for Histological Bone Samples
This is to preserve tissue samples permanently in as life-like state as possible and to allow for the preparation of thin, stained sections for histological analysis.
Microbiological Studies and Analysis
MCBLP-001MC: Bacterial Growth Curve Using 96-Well Plate
This is to measure the growth of bacteria using a 96-well plate instead of the classic cuvette method at OD600. The protocol only requires 100 μL of media to obtain the growth curve.
MCBLP-002MC: Bacterial Biofilm Formation Assay using a 96-Well Plate
To quantify the level of bacterial biofilm formation using a spectrophotometric method.