Haemocytometer

There are two methods of estimating the number of cells in a suspension. Using a haemocytometer the number of cells in a given volume is counted by direct microscopic examination. Using electronic counters, e.g. the Coulter counter, the cells in a given volume of suspension are drawn through an orifice and registered electronically. 

A drop of cell suspension (containing between 105 and 106 cells/ml) is placed at the two edges of the coverslip so that the suspension flows into the chambers by capillary action (Fig. 7.1). Do not flood the chamber. The slide is viewed under a low power objective of a microscope. 

The cells in four large squares in the comers of each of the two ruled areas are counted. Count cells touching the right and top lines but not those touching the left and bottom lines. The volume of a large square is 0.1 mm3 and the average cell count should therefore be multiplied by 104 to give the number of cells per ml. 

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Mix 10 pil cell suspension with 10 p.1 ofTtypan Blue solution and estimate the viable cell density using a haemocytometer. Non-viable cells turn blue. 

Sf9 cell culture 150-mm tissue culture plates High-titre recombinant viral stock EX-CEL 405 serum-free medium containing 50 xg/ml gentamycin sulphate, 2.5 i.g/ml amphotericin B, and 10% fetal calf serum 27°C incubator Microscope Haemocytometer... 

Calculate the cell density by diluting 10 pL of suspension in 90 pL of nigrosin then count the cells using a haemocytometer. Adjust the density to 5 x 105 cells/mL in production medium and inoculate the reactor with 35 mL of this suspension using the following procedure (see Note 12). 

Assessment of the stability of an emulsion against coalescence involves droplet counting218. The most unequivocal method (but one which is rather laborious) is to introduce a suitably diluted sample of the emulsion into a haemocytometer cell and count the microscopically visible particles manually. 

Take up some trypan blue cell suspension in a Pasteur pipette and fill a haemocytometer counting chamber by capillary attraction ( 7.2.1). Take care not to flood the channels of the chamber. Count the cells under X10 objective. 

Fig. 7.1. Use of the haemocytometer. The diagram on the left shows a modified Neubauer haemocytometer and on the right is a photomicrograph of BHK21C13 cells...

Discard the supernatant and resuspend the cell pellet in 1 mL of HBSS/BSA. Mix 10 ul. of the cell suspension with 90 pi. of Kimura stain. With this stain, eosinophils have a green cytoplasm while mononuclear cells and neutrophils do not (all cells have a blue nucleus). Count the different cell types in a haemocytometer. Eosinophil purity should be >95% the predominant contaminating cells are mononuclear and the major exclusion criterion for the majority of our studies is the presence of >1% neutrophils. 

Removal of cryopreserved cells from storage Resuscitation of cryopreserved cells and their first subculture Use of haemocytometers for the calculation of cell densities... 

To measure performance, to achieve reproducibility or to make comparative studies, a means of quantifying the cell population is needed. Classically, direct counts of cell numbers using a microscopic counting chamber (haemocytometer), usually in conjunction with a vital stain (e.g. Trypan blue) to distinguish viable and non-viable cells, is used. However, all vital stains are subjective and cannot give absolute values, and cell numbers take no account of differences in cell size/mass. The method is simple, quick and cheap, and requires only a small fraction of the total cells from a cell suspension. 

The most common routine method for cell counting that is efficient and accurate is with the use of a haemocytometer. There are several types on the market, of which the Improved Neubauer has proved most popular. 

Figure 2.2.1 Improved Neubauer haemocytometer with coverslip.

Haemocytometer with coverslip - improved Neubauer British Standard for Haemocytometer Counting Chambers, BS 748 1963 (Figure 2.2.1)... 

Thoroughly clean the haemocytometer and coverslip and wipe both with 70% alcohol before use. 

The following can cause inaccuracy of the haemocytometer method by affecting the volume of the chamber ... 

For direct cell counting, pool eight replicate wells and carry out counts using haemocytometer and Coulter counter. 

Plate cells at 1 x 10, 5 x 1(P, 1 x 10 and 5 x 10 cells mH in 7.5%, 5%, 2% and 1% serum for each of the test serum lots. Use the nutrient medium the cells have been growing in and plate 5 ml in each 60-mm Petri dish. Grow the cells for 10-12 days in a humidified (>95%) 5% 2/95% air atmosphere. Collect all the cells (trypsinization or centrifugation) from the plates and count by haemocytometer or Coulter counter. Plot growth curves for each of the serum concentrations. Select the combination with the most cell doubling at the lowest serum concentration. This will select the serum lot with fewest inhibitory factors. 

During the culture, the concentrations of living and dead cells (by the Trypan blue exclusion method) were measured using a haemocytometer (Chapter 2, section 2.2) glucose, lactate and glutamine by enzymic methods ammonia with a selective electrode and monoclonal antibodies by ELISA assay. 

Monitor cell growth at least daily by taking a small sample from the side arm (remove flask to a tissue culture cabinet) and carrying out a cell count (Trypan blue stain and a haemocytometer). 

Direct microscopic counting (using Helberor haemocytometer counting chambers)... 

Flocculation was measured by an improved Neubauer model of haemocytometer. The number of globules with respect to time was counted with the help of a hand tally counter (Erma, Tokyo) under the Olympus microscope. 

The rate of coalescence of the droplets in a macroemulsion is stated to be the only quantitative measure of its stability (Boyd, 1972). It can be measured by counting the number of droplets per unit volume of the emulsion as a function of time in a haemocytometer cell under a microscope (Sherman, 1968) or by means of a Coulter centrifugal photosedimentometer (Groves, 1964 Freshwater, 1966). 

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