Cell Culture Protocols 3 & 4 PDF

Protocol 3 - Cell Quantification Aim For the majority of manipulations using cell cultures, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. Using a consistent number of cells will maintain optimum...

Protocol 3 - Cell Quantification Aim For the majority of manipulations using cell cultures, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. Using a consistent number of cells will maintain optimum growth and also help to standardise procedures using cell cultures. This in turn gives results with better reproducibility. Equipment Personal protective equipment (sterile gloves, laboratory coat, safety visor) Waterbath set to appropriate temperature Microbiological safety cabinet at appropriate containment level Centrifuge CO2 incubator Haemocytometer Inverted phase contrast microscope Pre-labelled flasks Materials Media– pre-warmed to appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and temperature) 70% (v/v) alcohol in sterile water 0.4% Trypan Blue Solution Trypsin/EDTA Procedure 1. Bring adherent cells into suspension using trypsin/EDTA as described previously (Protocol 2) and resuspend in a volume of fresh medium at least equivalent to the volume of trypsin. For cells that grow in clumps centrifuge and resuspend in a small volume and gently pipette to break up clumps. 2. Under sterile conditions remove 100-200μl of cell suspension. 3. Add an equal volume of Trypan Blue (dilution factor =2) and mix by gentle pipetting. 4. Clean the haemocytometer. 5. Moisten the coverslip with water or exhaled breath. Slide the coverslip over the chamber back and forth using slight pressure until Newton’s refraction rings appear (Newton’s refraction rings are seen as rainbow-like rings under the coverslip). 6. Fill both sides of the chamber with cell suspension (approximately 5-10μl) and view under an inverted phase contrast microscope using x20 magnification. SigmaAldrich.com/ecacc 45 7. Count the number of viable (seen as bright cells) and non-viable cells (stained blue). Ideally >100 cells should be counted in order to increase the accuracy of the cell count (see notes below). Note the number of squares counted to obtain your count of >100. 8. Calculate the concentration of viable and non-viable cells and the percentage of viable cells using the equations below: Protocol 4 - Cryopreservation of Cell Lines Aim The protocol below describes the use of passive methods involving an electric -80°C freezer for the cryopreservation of cell cultures. ECACC routinely use a programmable rate controlled freezer. This is the most reliable and reproducible way to freeze cells but as the cost of such equipment is beyond the majority of research laboratories the methods below are described in detail. If large numbers of cell cultures are regularly being frozen then a programmable rate controlled freezer is recommended. Materials Freeze medium (commonly 90% FBS, 10% DMSO or glycerol) 70% (v/v) alcohol in sterile water PBS without Ca2+/Mg2+ 0.05% trypsin/EDTA in HBSS, without Ca2+/Mg2+ DMSO Equipment Personal protective equipment (sterile gloves, laboratory coat) Full-face protective mask/visor Waterbath set to appropriate temperature Centrifuge Microbiological safety cabinet at appropriate containment level Haemocytometer or automated cell counter like Muse® Cell Analyzer or Scepter™ Cell Counter Pre-labelled ampoules/cryotubes Cell Freezing Device Procedure 1. View cultures using an inverted microscope to assess the degree of cell density and confirm the absence of bacterial and fungal contaminants. Harvest cells in the log phase of growth. For adherent cell lines harvest cells as close to 80 - 90% confluency as possible. 2. Bring adherent cells into suspension using trypsin/EDTA as described previously (Protocol 2– Subculture of adherent cell lines) and re-suspend in a volume of fresh medium at least equivalent to the volume of trypsin. 3. Remove a small aliquot of cells (100-200μl) and perform a cell count (Protocol 3 – Cell Quantification). Ideally, the cell viability should be in excess of 90% in order to achieve a good recovery after freezing. 4. Centrifuge the remaining culture at 150 x g for 5 minutes. 5. Re-suspend cells at a concentration of 2-4x106 cells per ml in freeze medium. 6. Pipette 1ml aliquots of cells into cyroprotective ampoules that have been labelled with the cell line name, passage number, lot number, cell concentration and date. 7. Place ampoules inside a passive freezer e.g. Nalgene Mr Frosty box (Sigma-Aldrich cat no. C1562.) Fill freezer with isopropyl alcohol and place at -80°C overnight. 8. Frozen ampoules should be transferred to the vapour phase of a liquid nitrogen storage vessel and the locations recorded.