细胞培养常规操作

常规操作(主要内容如下)

・ Aseptic Technique

・ Culture Vessels

・ Cell Counting

・ Primary Culture

・ Maintenance of Cell Line

・ Hybridoma

・ Cell harvesting

・ Spcial Cell and Tissure Culture

・ Antibiotics and contamination

・ Others

Aseptic Technique

・ Aseptic Technique (Contributed by Nanci Donacki)
Aseptic techniques used by Cell Culture specialists in handling products from and/or mammalian cells.

Culture Vessels

・ Recommended working medium, trypsin volume and cell inoculation density
Useful data for cell subculture with different culture vessels.

・ Useful Numbers for Cell Culture (LTI)
Reference table of usr/localious culture vessels and their area, planting cell number, cell yield and more

• Surface Areas of Cell Culture Vessels (LTI)
  

・ Plastic Dishes for Growing Cells (Sefton Lab)

・ Working Volumes for Tissue-Culture Vessels (Becton Dickinson)
A comprehensive chart for determining appropriate volumes of medium, wash buffers and trypsin for specific tissue culture vessel configurations)

Cell Counting

• Use of a Hemacytometer ( P.J. Hansen Lab)
  Very detailed guide on counting cells using a hemacytometer

・ Hemacytometer Reference Guide (Becton Dickinson)
Illustrated instruction for using a hemacytometer to count cells

・ Eukaryote Growth Dynamics (William H. Heidcamp)
For determining cell doubling time.

・ Cell Counts Using a Hemocytometer (Donis-Keller lab)
The purpose of this procedure is to determine the cell density of the culture. Cell cultures always have some dead cells; the viable and non-viable cells can be distinguished with the use of trypan blue dye and a hemacytometer. Living cells will not take up the dye while dead cells do.

・ Cell Counting (Sefton Lab)

・ Viable Cell Counts Using Trypan Blue (LTI)
Viable Cell Counts Using Trypan Blue Viable Cell Counts Using Trypan BlueTrypan blue is a vital dye. The reactivity of trypan blue is based on the fact that the chromopore is negatively charged and d

・ Viability Cell Count (William H. Heidcamp)

Primary Culture

・ Establishment of a Primary Culture (William H. Heidcamp)

・ Culture of Endometrial Explants (P.J. Hansen Lab)
This method is used to study secretion of proteins and prostaglandins by endometrium from the cow, ewe, mare, bitch and other species. The technique is also useful for culture of peri-implantation conceptuses and placental tissues for metabolic labelling studies and to obtain conceptus secretory proteins for biological studies.

・ Fractionation of Cell Types (LTI)
Separation of blood or tumor cells into different cell types on density gradients is done frequently for establishment of primary cultures. In a cell suspension containing a mixed population of cells, cells can be separated on the basis of both size and density.

・ Dissociation of cells from Primary Tissue (LTI)
Several methods are introduced.

Maintenance of Cell Line

・ Maintenance of Cell Culture
Detailed procedure for culture and subculture cell in flask and plates

・ Adaptation of Cells to Serum Free Medium (LTI)
Adaptation of Cells to Serum Free Medium Adaptation of Cells to Serum Free Medium SEQUENTIAL ADAPTATION Subculture the cells growing in serum-supplemented medium into a 1:1 (v/v) mixture of SFM and...

・ Lymphocyte Transformation (Donis-Keller lab)
Lymphocytes are transformed to establish cell lines. Mononuclear cells (lymphocytes) from anticoagulated venous blood are isolated by layering onto histopaque. During centrifugation, erythrocytes and granulocytes are aggregated by ficoll and rapidly settle to the bottom of the tube; lymphocytes and other mononuclear cells remain at the plasma-histopaque interface. Erythrocyte contamination is neglible. Most extraneous platelets are removed by low speed centrifugation during the washing steps.

・ Preparation of Lymphoblastoid Cell Lines for Long-Term Storage (Donis-Keller lab)
To store cell lines in a form that will insure recovery with high viability. A culture in logarithmic phase of growth with a total volume of 80-100 ml/T-75 flask should yield enough cells to freeze 10 ampules (1.0 ml/ampule). Cells should have a count of 4 X 106 cells/ampule to 9 X 106 cells/ampule. Too high or too low a cell count lowers recovery viability. Cell are frozen in RPMI-1640 with 15% Fetal Bovine Serum + 10% DMSO. Cultures are frozen slowly using a Model 700 Controller freezing chamber. This precision electronic device automatically controls the injection of liquid nitrogen into the freezing chamber to provide a 1 degrees C/minute freezing rate from +4 degrees C to -45 degrees C (with automatic heat of fusion compensation), then a 10 degrees C per minute freezing rate to -90 degrees C. Frozen ampules should be stored in liquid nitrogen for long term storage or in a -135 degrees C Cryopreservation System. Note: Cryotubes should be labeled with cell line number anddate prior to beginning this procedure.

・ Maintaining Lymphoblastoid Cell Lines (Donis-Keller lab)
To grow lymphoblastoid cells for permanent storage and for DNA extraction.

・ Lymphoblastoid Cell Lines from Frozen Whole Blood (Donis-Keller lab)
Blood Samples can be stored frozen as a backup in case an LCL is needed at a later date.

・ Cell Suspension Culture (Gimila's Lab)
Tobacco cell culture method including media preparation

・ Transferring CHO Cells From Monolayer to Suspension Culture (LTI)

・ Transfer of Eukaryote Suspension Cultures (William H. Heidcamp)

・ Culture of BEND Cells (Bovine Endometrial Cells) (Peter J. Hansen)

・ Culture of endometrial explants (Peter J. Hansen)

Hybridoma

・ Cloning by Limiting Dilution (Contributed by Nanci Donacki)

Cell harvesting

・ Non-enzymatic Methods for Cell Harvesting (NUNC)

・ Preparation of Lymphocyte Cell Pellet for Storage (Donis-Keller lab)
Following propagation to 1 X 108 cells, lymphoblastoid cells are conveniently stored at -80 degrees C to preserve the high molecular weight DNA in the cells until the DNA is purified. This procedure describes the steps required to harvest and freeze the cells for long term storage.

・ Trypsinizing Cells (Sefton Lab)

・ Dissociation of Cells from Culture Vessels (LTI)
Dissociation of Cells from Culture Vessels Dissociation of Cells from Culture Vessels The following is a general procedure to rapidly remove usr/localious cell lines from the substratum while maintaining...

・ Dissociation of Cells from Primary Tissue (LTI)
Dissociation of cells from Primary Tissue Dissociation of cells from Primary Tissue TRYPSIN After dissecting off unusable tissue, mince the remaining tissue into 3 to 4 mm pieces with a sterile...

• Trypsinization of Adherent Cells (Suprya Jayadev)

Spcial Cell and Tissure Culture

• Culture of endometrial explants (P.J Hansen Lab)
  
• Culture of BEND cells (a bovine endometrial cell line) (P.J Hansen Lab)
  
• Tissue Culture of PtK ₁ Cells (Salmon Lab, University of North Carolina at Chapel Hill, Department of Biology)

Antibiotics and Contamination

・ Decontamination of Cultures with Antibiotics (LTI)
Decontamination of Cultures with Antibiotics Decontamination of Cultures with Antibiotics When an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the...

・ Use of Antibiotics and Antimycotics (LTI)
provides a reference table of commonly used antibiotics with their concentration in media, antibiotic spectrum and stability

・ Removal of Yeast Contamination from Lymphoblast Cultures (Donis-Keller lab)
This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when slightly shaken and lymphocytes will not cluster together as much as normal. If cultures are suspect, a drop of culture can be streaked on a YPD media plate to check for growth of yeast colonies, or a 5 ml sample can be taken to Barnes Diagnostic Center for identification of yeast strain.

Others

・ Tobacco Cell Suspension Culture Methods (Gimila Lab)

・ Mini-Chamber for regulating gaseous environment during culture (P. J.Hansen Lab)

・ Logging In Specimens and Record Keeping (Donis-Keller lab)
To keep a written and computerized record of all cell lines, the dates when cell lines were received and frozen, freezer locations, and any other important information such as dates of birth, sex, etc.

• Infection Cell with Retroviruses (Sefton Lab)

• Checklist for Media Replacement (Becton Dickinson)
  Things to remember and prepare before feeding your cells

• Checklist for Subculturing (Becton Dickinson)
  Things to remember and prepare before splitting your cells into fresh tissue culture vessels
  
• Hemacytometer Workbook (Becton Dickinson)
  Sample problems for determining cell number, cell density, viability, etc.
  
• Preparing coated coverslips (Hoshi Lab)
  Some cells stick to coveslips better if they are coated with poly amino acids or collagen. Poly-(D, L, D/L)-lysine is commonly used to treat cover slips.
  
• Tips (Sefton Lab)
  How often do cells need to be transferred and at what density should they be seeded when they are transferred or passaged?
  
• Adaptation of Cells to Serum Free Medium (LTI)
  Adaptation of Cells to Serum Free Medium
  
• Troubleshooting: Cell Culture (LTI)
  Troubleshooting to commonly encountered problems.