Hemocytometer

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional (13) and sampling (14—16) errors. The distribution of cells across the surface of a hemocytometer is sensitive to the technique used to charge the hemocytometer, and nonuniform cell distribution causes counting errors. In contrast, flow cytometer counts are free of distributional errors. Statistically, count precision improves as the square root of the number of cells counted increases. Flow cytometer counts usually involve 100 times as many cells per sample as hemocytometer counts. Therefore, flow cytometry sampling imprecision is one-tenth that of hemocytometry. 

Aperture impedance and most other automated counters measure MCV and RBC independently, in contrast to the manual methods where MCV and MCH accuracies depend on hemocytometer red cell count accuracy. 

The cells (1-3 x 106 cclls/rnl) were incubated for 24 h at 37 °C in RPMI1640 medium supplemented with 8mM NaHC03, 20mM HEPES, 5% FCS, 10 pg streptomycin, and lOU/ml penicillin without agents or in the presence of fullerenes C60. The number of viable cells was counted in hemocytometer using 0.4% solution of trypan blue. 

To subculture cells, resuspend the cells by pipetting the cells up and down to break down cell clusters. Count the cells using a hemocytometer. 

For making frozen stocks, the cells should be counted using a hemocytometer. 

A hemocytometer can be used to estimate exactly the number of cells in a suspension. The hemocytometer consists of two chambers, each of which is divided into nine 1.0 mm squares (Fig. D.4). A cover glass is supported 0.1 mm over these squares such that the total volume over each square is 1.0 mm x 0.1 mm or 0.1 mm or lO cm . Since 1 cm is approximately equivalent to 1 ml, the cell concentration per ml will be the average count per square x lO. ... 

Place a cover glass over the hemocytometer chamber. 

The number of cells is counted in a counting chamber such as a hemocytometer. 

A miniaturized version of the conventional flask method with S. capricomutum has been developed by Blaise et al [136]. In this assay the algae are exposed to the toxicant in 96-well microplates for a period of 96 hours, after which the cell density is determined using a hemocytometer or electronic particle counter. ATP content measurements [136] or chlorophyll fluorescence [141,142] have also been proposed as test criteria. Compared to the flask method, the main advantages of the microplate assay are (a) the small sample volumes and reduced... 

Cell Culture 1. Detach cell monolayer and obtain a single cell suspension. Determine cell concentration using a hemocytometer. 2. Adjust the concentration to the appropriate cell density (here we used 0.5-2 xlO cells/mL), according to the method of choice, in order to obtain spheroids of at least 300 pm diameter (see Note 1). 

Detach HUVECs and obtain asingle cell suspension. Determine cell concentration using a hemocytometer. 

For determination of yield, mix 10 pi of the cell suspension with 10 pi Soln. E and count using an Neubauer ruling hemocytometer. 

Centrifuge exponentially growing rat or mouse myeloma cells in 50-inL aliquots for 3 min at 400g, wash twice by resuspension m serum-free DMEM, count in a hemocytometer, and resuspend in this medium to 1—2 x 107 cells/mL. 

Count viable lymphoid cells in a hemocytometer. Spleens from immune mice yield about 108 cells, from rats 3-5 x 108 cells, and the mesenteric nodes of rats up to 2 x 108 cells. 

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional and sampling errors. 

Neubauer hemocytometer (cat. no. MNK-420-010N Fisher Scientific UK, Bishop Meadow Road, Loughborough, Leicestershire, UK). 

A modified Neubauer hemocytometer was used at 40x magnification to obtain the number and size range of gel spheres in suspension. The depth of the counting chamber was increased from the usual 0.1 mm for routine cell counting to 0.3 mm to ensure unrestricted influx of the larger gel particles. A well-shaken 1 1 (v/v) suspension of pre-swelled gel spheres in PBS was diluted 5-fold and sampled with a 1-ml plastic serological pipet (Falcon, Oxnard,... 

Ca.). The pipet had been pre-coated by drawing and discharging a gel sphere suspension several times, followed by rinsing 3 times with PBS. The number of spheres counted in the hemocytometer was subsequently converted to concentration (number of spheres per ml). The size distribution of the hydrated gel spheres was determined by measuring the diameter of each sphere in the sample with the aid of the hemocytometer grid squares of known dimension. 

H. Leon Bradlow (Rockefeller University). Cells were grown in RPMI 1640 supplemented with 10% Fetal Calf Serum (Gibco) and pen/strep/ fungizone. Experiments were performed in microtiter wells or 35mm tissue culture dishes. Cells were enumerated using a hemocytometer. 

Cell concentration in suspension can be determined through an optical microscope employing a hemocytometer for manual cell counting, or in a semi-automatic way using an electronic particle counter (such as a Coulter counter), as described in detail by Freshney (2005). Through dye exclusion (such as trypan blue), it is possible to determine viable cell concentration, that is the number of cells in a known sample volume capable of proliferating in favorable culture conditions. 

When the deoxynucleotide is associated with a fluorochrome, the cells can be observed under a fluorescence microscope, whereby apoptotic cells present an intense fluorescence and, in advanced stages, nuclear fragmentation can be visualized. For a quantitative analysis, either a hemocytometer in an optical microscope or a flow cytometer can be used (Tinto et al., 2002). Peroxidase-marked deoxynucleotides can be quantified by chromo-genic tests that use the enzyme substrate. Indirect methods use antigens linked to the nucleotide and recognized by labeled antibodies. However,... 

For microbial cultures it is common to express cell concentration as dry cell weight by volume, in animal cell cultures the number of cells per unit volume is more commonly used (for instance, 1.3 X 106 cells/ml). For the determination of cell number a microscope graduated slide can be used such as a hemocytometer or Neubauer chamber. In these, the thickness between the slide and the coverslip is fixed and known (typically 0.1 mm) and graduation marks on the slide allow the number of cells to be counted in a certain area. The volume can be determined by multiplying this area by the known thickness. With the aid of a dye (such as trypan blue), it is possible to estimate cell viability. This is expressed as the percentage of viable cells determined as those with an intact membrane capable of excluding the dye from inside the cell and so remaining colorless (see Chapter 2 and Freshney, 2005). 

Resuspend the cells in F/20 medium, 4 mL/femur and count the nucleated cells in a hemocytometer after staining with methylene blue, or count the cells on an electronic cell counter after first lysing the red cells with Zapoglobin. Each femur will yield 10-15 x 106cells. 

C. Hepatocytes are counted in a hemocytometer and viability is determined using the trypan blue exclusion method. Viability is always > 90 % with a yield of 2-3 x 10s cells. Cells are resuspended in medium and diluted to a final concteration of 1 x 106 cells/ml. 

Tumor cells are detached by EDTA treatment, rinsed with PBS and diluted in PBS at approximately 10 /ml. 100 /rl of the cell suspension are plated onto platelets and incubated for 1 h at 37 C. After washing three times with PBS, adherent tumor cells are removed by trypsin/EDTA treatment, and counted with an hemocytometer under phase microscopy. Alternatively, tumor cells can be radioac-... 

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