Haemocytometer cell count

A Trypan blue is harmful if ingested or inhaled. It is irritating to the eyes, harmful 

Appropriate precautions should be taken when handling Trypan blue and the use 

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

Moisten the edges of the coverslip or breathe on the chamber to provide moisture before placing the coverslip centrally over the counting area and across the grooves. 

Gently move the covershp back and forth over the chamber until Newton s rings (rainbow-hke interference patterns) appear, indicating that the coverslip 

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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. 

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. 

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

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). 

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). 

After the phototaxis plates have been inoculated, determine the cell counts that were used by using a haemocytometer to count cells in the separate counting dilution that was made earlier. This means that exact densities are not controlled beforehand but are measured after the plates have been set up. However, the standard nature of the procedure with the use of the packed cell pellet as the dense suspension ensures that the densities are in approximately the required range. 

Fig. 3. Effect of time of addition of 5-methylnicotinamide on T. cruzi differentiation. Amastigotes were transferred to Warren s medium at iTC at time zero. 5-Methylnicotinamide was added, to a final concentration of 5 mM, to separate cultures at the times indicated and the numbers of flageh lated cells counted with a haemocytometer at 96 h. This was expressed as a percentage of the flagellated cells in control cultures without 5-methylnicotinamide. Data taken from [8], by copyright permission of the Rockefeller University...

Lu et al. (1993) observed the interaction of DLC (obtained by IBAD) with human embryonic kidney (HEK-293) cells using a haemocytometer for cell counting and trypan blue dye exclusion to assess HEK-293 cell viability in DLC-coated P-35 dishes. According to Lu et al. (1993), HEK-293 cells grew well there was no delayed attachment to the DLC-coated dishes compared with the control, and both the cells growing in the DLC-coated and control dishes had a cell viability of 60% on the first day of incubation that increased to greater than 90% on the second day of incubation. 

At the end of the culture should a cell count be needed remove a measured aliquot of beads (e.g. 20 cc), add 100 ml of 0.1% citric acid and 0.2% Triton X-100, place on an orbital shaker (150 rev/min) at 37 C for 2 h. Collect supernatant, measure the volume, wash beads with PBS, add ciystal violet to the supernatant (0.1% final concentration), and cany out a nuclei count in a haemocytometer. 

Figure 4. Comparison of growth profiles over 96 h for recombinant BHK cells grown on poly(vinyl chloride) (PVC) and Control (TCP) surfaces. Cell counts for die PVC group were obtained by haemocytometer.

Figure 5. Dependence of cell counts after 24 h on serum (FBS) supplement level for recombinant BHK cells grown on Control (TCP) and Nation surfaces. All cell counts were obtained by 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). 

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. 

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. 

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. 

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. 

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. 

Centrifuge exponentially growing mouse or rat myeloma cells in 50 ml aliquots for 3 min at 400 g, wash twice by resuspension in serum-free DMEM, count in a haemocytometer, and resuspend in this medium to 1-2 X 10 cells/ml. 

Count viable lymphoid cells in haemocytometer. Spleens from immime mice yield about 10 cells, from rats 3-5 x 10 cells, and the mesenteric nodes of rats, up to 2 X 10 cells. 

Harvest cells and medium from wells, giving a slight tap to dislodge cells and get them in homogeneous suspension. Pool cells, cany out a haemocytometer count, and add to a 25 cm flask containing 7 ml pre-warmed mediiun at 2-5 x 10" cells/ml. Gas with 5% CO2 and incubate for a further four days. 

Harvest the culture, pool cells, carry out a haemocytometer count. The object is to store five to ten ampoules containing S x 10 cells per ampoule, leaving enough cells over to continue a new culture in a 7S or 175 cm flask. 

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