Index

1_ Introduction

• advantages of cell culture 1: Environmental control
• advantages of cell culture 2: Cellular homogeneity of the different batches
• advatanges of cell culture 3: other factors
• Limitation of cell cultures 1: Phenotypic and genotypic variability
• Limitations of cell culture 2: morphological and functional differences with cells in vivo

2_ layout of the cell culture laboratory and necessary equipment

• laminar-flow hood
• Laminar flow hood: selection and installation.
• Laminar flow hood: working tips
• Water bath: mainteinance and usage protocol to avoid contamination

3_ Aseptic technique

• Decontamination
• aseptic technique SOP
• A guide to cell-lab personal hygiene
• Other tips for sterile handling

4_Culture media

• Cell culture media history
• CO₂ independent media

4.1 Phisicochemical properties

• culture media pH
• Temperature in cell culture

4.2 the components of cell culture medium

• The components of the cell culture medium: HEPES
• The components of the cell culture medium: glutamine
• Phenol red

5_ Culture Vessels

• culture vessels: choice of substrate materials
• Glass slide for cell culture: prep protocol

6_ Media and reagents

• Media, reagents, and solution preparation
• Preparation of cell culture media
• PBS recipe and preparation
• Protocol: 4% Paraformaldehyde solution in PBS
• Scott’s tap water substitute

7_Subculture

• Subculture of adherent cells.
• Trypsinization: protocol, tips and tricks
• The split ratio

8-Quantitation

• how to calculate the cell seeding density
• In how much volume resuspend the cells to plate them at the desired cell density?
• Cell counting
  • Cell counting: SRB Assay
  • Cell counting: hemocytometer
  • Coulter counter
  • cell quantitation: Crystal violet
  • Indirect cell numebr quantitation: protein content
    • Quantifying protein using absorbance at 280 nm
    • The Bradford method to quantify protein
    • The Lowry method to quantify protein
• Methods to estimate Cell Confluency

9_ Contamination

• Cell culture contamination: main causes
• Contaminated! When you see it by eye
• Contamination: when it is hard to detect – Mycoplasma
• Summary of contamination preventive methods
• Summary of Contamination Containment Methods
• Decontamination
• Eradication of fungus and mold contamination
• Eradication of bacteria contamination
• Eradication of mycoplasma contamination: antibiotics

Protocol: eradication of mycoplasma from cell culture