Cell count, 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. 

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. 

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,... 

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. 

Cell counting chamber (Hemocytometer or disposable Kova slides). 

Depending upon the test organism, between 2 X 104 and 5 X 104 cells are used to inoculate the test vessel and the concentration of cells is determined daily. Cell counts are made daily by using a hemocytometer or an electronic particle counter such as the Coulter Counter. Chlorophyll can be measured spectrophotometrically or fluorometrically. The fluorometric determinations are more accurate at low concentrations of test organism. Other measurements that have been used include DNA content, ATP charge, and 14C assimilation. 

Cell counting 0.4% (w/v) Trypan blue (Biocompare, South San Francisco, CA) and hemocytometer slides (Hausser Scientific, Horsham, PA) were used for cell counts. 

The first hematologic cell-counting chamber, the hemocytometer, was developed in 1873. A drop of a diluted suspension of blood cells was inserted by capillary action between a glass cover slip and the finely cross-hatched surface of a glass chamber. The blood cells were allowed to setde to a common focal plane. Then, using a microscope, the red blood cells (RBC) within the cross-hatched areas were counted. Eventually, technologists used the hemocytometer to count white blood cells (WBC) and platelets (PLT) as well, first destroying the red blood cells which predominated. Hemocytometers are still used on occasion. 

Automated Cell-Coimting Instruments. Until the mid-1950 s, cell counts were performed manually using a diluted fluid and a hemocytometer, and blood smears were viewed microscopically. Modern automated instruments can perform a complete blood count (CBC), which includes red blood cells, white blood cells, platelets, hemoglobin, hematocrit, and a five-part differential. This testing accounts for... 

Isolation of parenchymal cells from rat liver was performed according to Berry (1974) except that the collagenase-hyaluronidase solution was not recirculated. Cell count was determined using a hemocytometer, and viability was estimated by 0.2% trypan blue staining ... 

For cell count consistency between screens, we use an automated cell counter such as the Invitrogen Countess or the Bio-Rad TC20. Both count cells and determine their viability using trypan blue. Alternatively a hemocytometer can be used. 

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. ... 

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

Count cell concentration using a hemocytometer. Count 4,... 

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-... 

Harvest cells and count using a hemocytometer (see Note 2). Place aliquots of 1 x 106 cells into flow cytometer tubes. Wash once in ice-cold PBS and pour off the PBS, leaving the pellet with a small amount of fluid. Cells are pelleted at... 

Count OM-10.1 cells using trypan blue and a hemocytometer and collect by centrifugation sufficient cells to perform the assay (7.5 x 10s cells needed for each test well). 

Count the number of live cells (translucent trypan blue is excluded) and the number of dead cells (blue trypan blue is not excluded) using a hemocytometer. Calculate the percentage of live cells in the well... 

Fig. 1. Hemocytometer counting grid. The center square of the hemocytometer is divided into 16 smaller squares and is edged by triple lines. Count all cells within the triple lined square, but exclude any cells that fall on the triple lines. For accurate counts, aim to count between 30 and 300 cells in each square.

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