Calciummagnesium to minimize cell sticking discard pbs in waste flask 3 add 3 ml of trypsin edta solution. C2c12 cell culture, differentiation treatment, and crosslinking protocol. If you are passing cells for transfection, use dmem without antibiotics. Splitting cells everything must be done with sterile technique in the hood. A media alternative includes alphamem and dulbeccos modified mem i. Ensure trypsin covers the entire surface on which cells are adherent allow 23 minutes for trypsin to work. Protocol culture conditions the cells should be preferably passaged 23 days before before nucleofection nucleofection. Pdf this paper aims to discuss trypsin enzymes from structural, catalytic, physiological, and the effects of trypsin deficiency disorder. Tissue dissociationprimary cell isolation and cell harvesting are principal applications for enzymes in tissue culture research and cell biology studies. All trypsin solutions are prepared from porcine parvovirustested and. Add the prewarmed dissociation reagent such as trypsin or tryple to the side of the flask.
It contains 10% fetal bovine serum as a trypsin inhibitor and cell protection agent. Harvesting cells from corning cellstack culture chambers. Vero cells are derived from the kidney of an african green monkey, and are one of the more commonly used mammalian continuous cell lines in microbiology, and molecular and cell biology research. Trypsinization time varies depending on the cell type. Tryple select porcine trypsin tryple select was stored at 4c, room temperature, or 37c. Any and all media, supplements, and reagents must be sterilized by filtration through a 0. This procedure should be done with your best sterile technique used for tissue culture.
If detachment is low, place flask in incubator and inspect microscopically every 5 minutes neuro2a cells detach easily at room temperature, 23 minutes is sufficient. This latter technique was used when results for the betaactin gene were negative and is summarized in table 1. Hek293 cells human embryonal kidney cells hek293, atcc. Subculturing adherent cells thermo fisher scientific in. Remove and discard the wash solution from the culture vessel. Passagingsplitting cells tissue culture protocol 0801. N4 cells protocol for all reagents thaw and prewarm trypsin prewarm pbs prewarm required media prewarm gelatin dishflaskplate volume of gelatinpbs volume of trypsin volume of media 6 well plate 3ml 500l 4.
Cell culture guidelines the following is a general guideline for culturing of cell lines. This unit includes protocols for the growth and maintenance of vero cell lines in a research laboratory. Trypsin may be used to remove adherent cells from a culture surface. Add 3ml t75 or 6ml t150 of trypsin edta solution and incubate at 37 c and observe after 5mins. Before beginning spray hood down with 70% ethanol and wipe clean. Resuspend cell pellet with the complete and dispense into the culture flasks from step 1. Cell surface proteins are essential for the normal functioning of cells in culture. Introduction adherent cell lines must be disrupted from cell culture flasks in order for them to be added to the neutralization assay or used for transfection for the production of pseudoviruses. Although all cells eventually dissociated from the plastic, the time required for cells treated with tryple select remained constant while the time required for cells treated with porcine trypsin more than doubled after 2 days.
Sera for cell culture with robust cell linesexcellent value. All cell culture must be undertaken in microbiological safety cabinet using. Breseagen protocol human embryonic stem cell protocols. Breseagen protocol human embryonic stem cell protocols this material was cultured and frozen using bresagens protocols. Using pipette, add enough trypsin edta to cover the cells at the bottom of the flask. Cornings subculturing monolayer cell culture protocol. Cell growth protocol and differentiation treatment for the c2c12 cell line from. Trypsin concentration in 1x working solutions can range from 0. Add required amount of prewarmed cell culture media to fresh flask.
For a 75 cm2 flask, remove all but 10 ml culture medium. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. Cells should be nucleofected after reaching 8090% confluency. The cell line c2c12 is an immortal line of mouse skeletal myoblasts originally derived from satellite cells from the thigh muscle of a two month old female c3h mouse donor 70h after a crush injury yaffe and saxel, 1977. Passage secondary culture when it becomes confluent by repeating steps 1 to. To a t75 flask, add 2 ml of trypsin edta to the flask and observe for cell layer detachment under an inverted microscope. General details of cell culturing and subculturing. Use aseptic technique to prevent microbial contamination. Stable expression is hard to achieve in this cell line and cells must be continuously tested for expression. Do not allow cells to sit in dissociation media for more than 10 minutes. Hek293 cells should not be used for nucleofection after passage number 20.
Regardless of whether you are performing cell culture in a research lab or production facility, need a special or defined formulation, or are growing cell lines, primary cells, or stem cells. This represents the peak mitotic activity, and is the optimum point at which to harvest for chromosome studies. The cell line c2c12 is an immortal line of mouse skeletal myoblasts originally derived from. In cell culture, the way i understand it is that you. Higher cell densities may cause lower nucleofection efficiencies. The cell culture protocols below have been adapted from the following sources. Cells are most commonly removed from the culture substrate by treatment with trypsin or trypsin edta solutions. Thaw corning matrigel basement membrane matrix overnight by submerging the vial in ice in a 4c refrigerator before use. Corning matrigel basement membrane matrix for 3d culture in vitro.
Test samples were left on cell monolayers until all mdck cells appeared to dissociate. This protocol assumes that the cells are being cultured in t150 flasks e. Fundamental techniques in cell culture sigmaaldrich. Gibco tryple express enzyme is a highly purified, recombinant cell dissociation enzyme that replaces porcine trypsin. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Trypsinization is the process of cell dissociation using trypsin, a proteolytic enzyme which breaks down proteins, to dissociate adherent cells from the vessel in which they are being cultured. Basic techniques for mammalian cell tissue culture tritech. The following is a general guideline for culturing of cell lines. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment. Depending on the cell line culture vessel may be gently tapped on the side of the flask. Gibco tryple express enzyme is a highly purified, recombinant celldissociation enzyme that replaces porcine trypsin. All cell culture procedures must be conducted in a biosafety cabinet.
All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Tap bottom of plate on the countertop to dislodge cells. Passage interval cells should be passaged at 8090% confluency. Sera for cell culture with robust cell linesexcellent value for basic research lowendotoxin sera for general cell culture with. The a549 cell line grows easily and cell count doubling time is typically 2440 hours. Warm up the differentiation and cell culture growth media. When added to a cell culture, trypsin breaks down the proteins which enable the cells to adhere to the vessel. Removal of adherent cells from culture surface using trypsin. Dislodge cells from the flask substrate with a cell scraper. Including cell culture basics, equipment, protocols and troubleshooting. Trypsin treatment for disassociating cells from culture vessel. Protocol for trypsin edta treatment for disruption of cell monolayers november 2018 i. Trypsin acts by cutting amino acids, specifically lysines or arginies, on their ctermini unless these amino acids are followed by an proline. Not all cells will require trypsinization, and to some cells it can be toxic.
Stable at room temperatureno need to thaw gentle on cells help protect your cells surface proteins figure 4. Wicell recommends that stem cells should be thawed and established in the conditions in which they were initially frozen prior to transfer to alternate culture platforms. Despite the widespread use of enzymes for these applications over the years, their mechanisms of action in dissociation and. Obtain a new flask for your cell culture and label it with the date and your initials and the cell type obtain your original culture flask from the incubator and place in the. Despite the widespread use of enzymes for these applications over the years, their mechanisms of action in dissociation and harvesting are not well understood. The epitopes at the cell surface are important in techniques that leverage them, such as phenotyping cells in flow cytometry. Gibco media, sera, supplements, cells, and cell culture reagents are designed to deliver reproducibility and performance for results you count on. Gibco trypsin solutions are used widely for dissociation of tissues and cell monolayers.
General protocol for recovering or freezing primary cells. B958 marmoset where a proportion of cells do not attach to the tissue culture flask and remain in suspension. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Protocol for trypsinedta treatment for disruption of cell monolayers. Protocol for trypsinedta treatment for disruption of cell. Add enough 37c trypsinedta solution to culture to cover adhering cell layer. To evaluate the extraction efficiency of genetic material from sera, trypsin and cell culture samples from different species we used realtime pcr for the betaactin gene and a conventional pcr protocol to detect the gapdh gene, which was developed in the present work. If passing cells for transfection, make sure there are not too many cells.
Corning matrigel basement membrane matrix for 3d culture. Chromosome harvesting from whole blood phytohemagglutinin pha, a lectin derived from the red kidney bean, is a powerful mitogen for human t cells 16. Culture guidelines of adherent cells using trypsin. Adherent cell lines must be disrupted from cell culture flasks in order for them to be. Pipette trypsinedta onto the washed cell monolayer using 1ml per 25cm2.
Make sure all materials are available before starting solutions, bottles, etc. Check culture with an inverted microscope to be sure that cells are rounded up and detached from. Notes hek cells are loosely adherent, and do not require digestion by trypsin edta prior to being passaged. The assay allows convenient testing of chymotrypsinlike, trypsin like or caspaselike proteasome activities using samples derived from cells in culture figure 5. Notes the amount of medium can be decided by the size of the cell culture dishes. Detection of contaminants in cell cultures, sera and trypsin.
Mammalian cell tissue culture techniques protocol abcam. Fibroblast subculturing protocol in cell culture, fibroblasts should be grown in 90% rpmi 1640 medium with 10% fbs added. Trypsinization is often used to pass cells to a new vessel. The medium contains unattached cells that may attach and grow, thereby providing a backup culture. Trypsin neutralization solution tns is a sterile, phosphate and hepesbuffered saline solution used to neutralize the effects of trypsin edta solution te. They can directly substitute for trypsin without protocol changes, and are. While the studies which require cell surface protein integrity, lower concentrated 0. Hek cells are loosely adherent, and do not require digestion by trypsinedta prior to being passaged. At the times indicated, samples were tested for the ability to cleave acetyl arginine pnitroaniline acargpna.
Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Therefore to maintain this heterogeneity both the attached cells and the cells in suspension must be subcultured. A549 cells are cultured in complete media consisting of dulbeccos mem modified with 10% fbs. Rinse the flask with sterile 1x pbs to remove all complete medium.
Stable at room temperatureno need to thaw gentle on cells help protect your cells. This protocol covers some basic techniques and suggestions for harvesting cells from cellstack culture chambers. The coupledenzyme format and speed of the proteasomeglo assay result in. Trypsin neutralization solution tns is a sterile, phosphate and hepesbuffered saline solution used to neutralize the effects of trypsinedta solution te. Hek cells are easy to culture and transfect, and contain the sv40 large tantigen, which allows for the.
The time of trypsin exposure should be kept to a minimum. Aspirate cell suspension and transfer to a conical tube. Examine the cells to ensure the cells are healthy and free of contamination. Once matrigel matrix is thawed, swirl vial to ensure the material is dispersed. Trypsin can also induce temporary internalization of some membrane proteins, which should be taken into consideration when planning experiments. These protocols are examples of methods for general cell culture, and have not been rigorously validated and optimized by qiagen. Cell growth protocol and differentiation treatment for the. On day 0, dilute the matrigel matrix to 5 mgml with icecold mdck complete cell culture medium. Katherine fisheraylor and brian williams c2c12 cell culture, differentiation treatment, and crosslinking protocol. Aspirate medium and wash the culture with pbs washing with pbs allows you to remove serum from culture conditions the serum proteins inhibit trypsin. Cell culture splitting cells lenzmeier research laboratory page 1 of 1 protocol for splitting cells t75 flasks. Passagingsplitting cells tissue culture protocol 08012009 2 7. The cells can be grown as adherent or in suspension in vitro. Hek293 cells human embryonal kidney cells hek293, atcc cat.
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