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G. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers. uk 42 • Trypan blue (vital stain) Cell Culture Protocols Equipment • Personal protective equipment (sterile gloves, laboratory coat, safety visor) • Waterbath set to appropriate temperature • Microbiological safety cabinet at appropriate containment level • Incubator • Pre-labelled flasks • Inverted phase contrast microscope • Centrifuge • Haemocytometer • Marker Pen • Pipettes • Ampoule rack • Tissue Procedure 1.

3. Collect an ampoule of cells from liquid nitrogen storage wearing appropriate personal protective equipment and transfer to the laboratory in a container of liquid nitrogen or on dry ice. It is important to handle the ampoules with care: on rare occasions ampoules may explode on warming due to expansion of trapped residual liquid nitrogen. 4. In a microbiological safety cabinet, hold a tissue soaked in 70% alcohol around the cap of the frozen ampoule and turn the cap a quarter turn to release any residual liquid nitrogen that may be trapped.

Take a small sample (100-200µl) of the cells from the cell suspension and count the cells (Protocol 6 - Cell Quantification). Calculate cells/ ml and re-seed the desired number of cells into freshly prepared flasks, without centrifugation, just by diluting the cells. Refer to the data sheet supplied with the cell line for the recommended seeding density. 4. Repeat this every 2-3 days. Key Points 1. g. recombinant protein or growth factor) of interest retain the spent media for analysis. 7 12 7 Protocol 6 - Cell Quantification Quantification Harvest cells (as per protocols 3 & 4) Re-suspend cells in fresh medium Remove 100-200µl of cell suspension Add Trypan Blue (dilution factor x 2) Prepare haemocytometer with coverslip Fill chamber with cell suspension Count cells Calculate concentration (refer to procedure) 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.

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Advanced Instrumentation and Measurement Techniques (csni-r1997-33)

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