![]() High-resolution details are obtained using TEM ( right panel corresponds to inset in previous image). Align fluorescence and TEM photomicrographs in order to obtain overlaid CLEM images ( middle panel). Use fluorescence images to identify cells of interest in TEM ( left panel). Obtain serial ultrathin sections from these blocks. Cells and the gridded pattern will be transferred to the surface of the resin block upon removal of the coverslip. Observation of cells with light microscopy and registration of cell position 4. Process coverslips for TEM by embedding in epoxy resin. Seeding of cells on gridded coverslips 2. ![]() Acquire bright-field images and fluorescence confocal Z-stacks at different magnifications. Identify cells of interest ( arrows) in the gridded coverslip and note their position in the grid. Cell number should be adjusted to a final confluency of ~50 %. Fluorescence-labeled cells are grown on gridded coverslips in multi-well plates to facilitate their identification. Haemocytometer arrangement and dimensions.CLEM procedure for cell cultures. Count the cells within the large square and those crossing the edge on two out of the four sides.įigure 3. Counting system to ensure accuracy and consistency. Appearance of the haemocytometer grid visualised under the microscope.įigure 2. of Viable Cells Counted / Total Cells Counted (viable and dead) x 100 = % viable cellsĬell Counting Calculator (opens in new window)įigure 1. *dilution factor is usually 2 (1:1 dilution with trypan blue), but may need to further dilute (or concentrate) cell suspensions.ġ0 4 = conversion factor to convert 10-4ml to 1ml (refer to figure 3 to view a diagram of the arrangement and dimensions) To calculate cell concentration per ml:Īverage number of cells in one large square x dilution factor* x 10 4 It is advisable to count around 40 to 70 cells to obtain an accurate cell count - therefore it may be necessary to count more than one large corner square.ĩ. Count viable (live) and dead cells in one or more large corner squares and record cell counts.Ĩ. To aid accuracy and consistency of cell counts use counting system illustrated in figure 2.ħ. ![]() Dead cells stain blue and are non-refractile. Live cells appear colourless and bright (refractile) under phase contrast. Trypan Blue is a "vital stain" it is excluded from live cells. Visualise the haemocytometer grid under the microscope, refer to figure 1 for layout of grid. Pipette trypan blue/cell mix (approximately 10µl) at the edge of the cover-slip and allow to run under the cover slip.Ħ. ![]() mix 100µl trypan blue stain with 100 µl cell suspension.ĥ. Mix equal volumes of 0.4% trypan blue stain and a well mixed cell suspension (not too vigorous) e.g. Newton's refraction rings are seen as rainbow-like rings under the cover-slip.Ĥ. Look for "Newton's Rings" which indicate that the cover slip has adhered via suction to the haemocytometer. Moisten (with water or exhaled breath) and affix cover-slip to the haemocytometer.ģ. Ensure the cover-slip and haemocytometer are clean and grease-free (use alcohol to clean).Ģ. Inverted microscope (preferably phase contrast)ġ. 0.4% Trypan Blue stain (fresh & filtered) in phosphate buffered saline ![]() Haemocytometer plus a supply of cover-slips This can be avoided by performing a viable cell count and following the recommended seeding density. One of the most common reasons for the failure to establish cells in culture is due to using an incorrect viable cell seeding density at the time of resuscitation i.e. ![]()
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