Cell suspensions coulter counter

The cell titer of an exponentially growing culture of cells in CM 10 is determined with a Coulter counter. The cell suspension is centrifuged at 70 g for 5 min and the supernatant is reduced such that 3 ml contains approximately 5 x 106 cells (3-h treatment) or 2 x 106 (treatment more than 3 h). 

Cell titres are determined by diluting of the cell suspension in Isoton and counting an appropriate volume (usually 0.5 ml) with a Coulter counter. Two counts are made per suspension. 

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

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. 

Fig. 3.8. L929 cells were seeded in 100 ml GMEM with 10% calf serum at 3 cells per microcarrier bead onto 5 x10s Cl-(Cytodex 1), P(Biosilon) or G (Bioglas) beads. The cell number was estimated by releasing the cells with trypsin, briefly allowing the beads to settle and counting the cells with a Coulter counter. Difficulty was encountered releasing the cells from Cytodex, but cells attached only weakly to the glass and particularly to the plastic beads and many cells were free in suspension. The use of electronic cell counters is not recommended for counting cells released from microcarriers as the orifice occasionally becomes blocked by small microcarriers in the...

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. 

After the supernatant is decanted, the pellet is resuspended as before in a limited amount of buffer, pH 6.5, without ACD and combined into one tube (a total of 3-4 ml). A Coulter Counter Model S plus IV is used to count a 1 10 dilution of platelets in buffer. The platelets are then adjusted with the final suspension buffer to a concentration of 1.25 x 109 cells per milliliter. These platelets, as small aliquots, are stored in capped tubes at room temperature. As needed, these aliquots are used for specific experiments. 

It is important that the growth of cultures be measured and related to the rate of mineral oxidation, and, if possible, that the numbers of cells on the mineral surface and in suspension in the medium be determined. The growth of cultures on a mineral-containing medium cannot be followed by turbidity or Coulter Counter measurements due to interference from the mineral particles. Microscopic counting has been used to evaluate bacterial numbers in the supernatant solution, but cells attached to the mineral surfaces cannot be counted nor can active and inactive cells be differentiated. The most-probable-number... 

Schwan (1957) wrote an introduction to impedance measurement on cell and bacteria suspensions. A Coulter counter is a streaming blood cell counter. Two different electrolyte reservoirs equipped with impedance CC electrodes are connected via a short capillary. The reservoir with the cell suspension is on higher pressure so that the electrolyte with cells stream through the capillary. Measured impedance is dominated by the capillary because of its very small diameter. At passage, the impedance goes through a peak because of the cell membrane. The frequency of the peaks is the number of cells passed per second the impedance waveforms are characteristic for different cell types. The cell suspension must have a concentration so that the probability of two cells in the capillary at the same time is low. The electrolyte concentration must be adapted to the impedance increase caused by each cell. 

The principle of the Coulter counter is based on letting cells in suspension pass a narrow orifice. If a cell has different electrical properties than the liquid, the impedance of the pore will change at each cell passage. Cell counting is possible, and it is also possible to have information about each cells size, form, or electrical properties. Figure 10.27 shows the basic setup of the two-electrode conductance measuring cell. Typical dimensions (diameter, length) for a capillary is 50 and 60 pm (erythrocytes) 100 and 75 pm (leukocytes). 

Pass the cell suspension over a sieve and centrifuge the filtrate at 200g for 5 min. Resuspend the pellet in 10 mL of medium and determine cell number using a hemocytometer or Coulter counter. 

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