Protocol for Poly-D-Lysine Preparation and Plate Coating

1. Prepare Poly-D-Lysine a. Add 100 ml of sterile tissue culture grade H2O to 5 mg poly-D-lysine (Sigma P6407). Final concentration of poly-D-lysine is 50 ug/ml. b. Mix by pipetting several times. c. Store at 2-8 ºC or –20 ºC. 2. Prepare Coated Plates a. Add enough poly-D-lysine solution to cover culture plate surface. b. […]

Cell trypsinisation

1. Remove medium from culture dish. 2. Gently wash cells in a PBS without Ca or Mg. 3. Remove the wash solution. 4. Add trypsin-EDTA solution to cover the bottom of the culture dish (2ml for 10cm dish, 0.5ml for 60mm dish). 5. Put dish in the 37°C incubator for up to 5 minutes. 6. […]

Heat inactivating serum

1. Place bottles in a 56oC water bath. Make sure the bottles cannot tip over or otherwise become submerged in the water bath. The temperature is critical for complement degredation, so make sure! 2. Wait until the temperature stabilizes at 56oC (after adding the serum to the water bath) 3. Gently swirl the bottles every […]

The basics about cell culture methods

The Department of Biological sciences of the University of Maryland (UMBC) has a protocol section very useful for cell culture first time users. They explain extensively all the components of the work area and the equipment mainly used, from how the hood works to the microscope or the incubators. Also, types of cells that grow […]

Fundamental techniques in cell culture

Sigma-Aldrich publishes in its learning center web site a very complete guide to cell culture techniques. It goes from the basic techniques to sterile techniques, freeze and thaw cells, culturing of semi-adherent or suspension cell lines, test for bacteria or fungi in the cultures, detection of mycoplasma,… In summary, an extensive guide for cell culture […]