Automated cell washing

Step 2. Instrumental isolation out of liquid culture media of individual cells. Step 3. Automated cell washing and cell suspension normalization to standard... 

Some of the problems surrounding the removal of glycerol from the high glycerol preparation have been overcome through the use of cell washing devices. One of these, developed by the Haemonetics Company (Braintree, MA) and based on the Cohn Fractionator, performs a continuous flow wash in a disposable bowl. The other apparatus developed by the IBM Corporation, conducts an automated batch wash in a disposable bag. Although effective. 

Harvest the cells from a sufficient number of tissue culture flaslcs. Wash the cells and resuspend in cold 1% BSA/PBS. If necessary break up clumps of cells by repeated pipetting or by syringing them through a 23G needle. Measure the cell concentration with a haemocytometer or automated cell counter and dilute to a final concentration of 4 x 10 /ml in cold 1% BSA/PBS. 

In an alternative procedure (84), the electrolyte is pumped through the cells at such a rate that the outlet concentration is 50 g/L MnSO and 67 g/L H2SO4. This spent electrolyte is then mixed with equal parts of make-up solution containing 150 g/L MnSO and the mixture returned to the electrolysis step. The electrolysis is continued over a period of days and terrninated when the EMD layer deposited on the anode reaches a specific thickness, usually on the order of 1—3 or 6—8 mm. Following completion of the electrolysis cycle, the entire electrode assembly is removed from the cell for removal of the deposited EMD, either manually or by an automated system (85). The product is repeatedly washed with water to extract the occluded acid (83) and dried at about 85°C in air. 

COS-7 or CHO cells (for initial transfection screening) or cells of therapeutic interest (e.g., dendritic cells and various cancer cells) at a confluence of 50%, grown in 96-well culture plates, were placed into the robot by the robotic conveyor. In a fully automated process, the robot removes the lid from the cell culture microtiter plate, dispenses lipoplexes into the wells (triplicates), replaces the lid and returns the plate to the incubator. After four hours, the cells are automatically retrieved, the cell monolayers are carefully washed using a special drop mode of the integrated plate washer, fresh medium is added, and the cells are incubated for further 42 hours before harvesting. 

An alternative method which can also be automated by the use of the Titertek supernatant harvester (see Appendix 3) involves the measurement of radioactive chromium released into the culture medium from killed cells. The harvester consists of a set of absorbent cylinders aligned so that they may be inserted into the wells of a microtitration plate (Appendix 3). Once the supernatant in the wells has been absorbed the cylinders are transferred to counting vials and the amount of radioactive chromium released from the cell monolayer is estimated. Cells take up 51 Cr sodium chromate rapidly and the excess is readily washed away by rinsing in culture medium. 

In the neutral red (cell viability) and total protein (cell proliferation) assays, cells are treated with various concentrations of a test substance in petri or multiwell dishes after a period of exposure, the substance is washed out of the medium. (An analytical reagent is added in the case of protein measurements.) Neutral red is a supravital dye, which accumulates in the lysosomes of viable, uninjured cells, and it can be washed out of cells, which have been damaged. In the protein test, Kenacid blue is added and reacts with cellular protein. Controlled cells are dark blue killed cells are lighter colored. The IC50 (the concentration which inhibits by 50%) is determined the test can be rapidly performed with automation. However, materials must be solubilized into the aqueous cell media for analysis. For many test materials this will require large dilutions which eliminate properties of the materials which cause irritation. 

Multiplicities of infection (MOI) of 10 1 and 2x10 1 (Salmonella THP-1 cells) were used. Salmonella cells were harvested at 3500 rpm for 8 min, washed 3 times in PBS and resuspended in 200 pL fresh antibiotic free RPMI medium. Bacteria and THP-1 cells were incubated together for 40 min at 37 C and were then decanted into sterile tubes and washed twice in pre-warmed PBS. Colistin was added at 50 pg/mL to inactivate extracellular bacteria and 200 pL of the cultures were placed into a black, clear-bottomed 96 well-plate. 0.1% saponin was added at time 180 min where appropriate. Imx and lux cultures of S. Typhimurium DT104 were used as controls. Bioluminescence was measured over 24 hours in an automated luminometer (Anthos) at 37 °C. 

Following the measurement, the beads are washed from the cell, which is then cleaned and made ready for the next sample. Automated instruments for clinical diagnostics, capable of handling multiple samples without operator intervention, are available. 

Specifically for Lab-Teks, we apply 250 p of the corresponding antibody by carefully distributing the fluid over the spotted area. We incubate for 10 min with the lid closed followed by 2 washes with 2 ml PBS (30 min each). We routinely include reliable stains highlighting cell nuclei (e.g., Hoechst or Dapi) which facilitates automated focusing and image acquisition (Liebel et al., 2003). The stained samples are stored at 4° either embedded in Mowiol or in PBS solution containing azide after a brief poststaining fixation of the samples with paraformaldehyde for 2 min. 

Figure 4.18 Schematic of a state-of-the-art apparatus for investigating the filtration, displacement washing and gas deliquoring phases of the filter cycle. (1) suspension feed vessel (2) wash liquor feed vessel (3) filter cell (4) rotary index table (5) electronic balance (6) pressure regulator. The inset photograph shows fully automated apparatus for obtaining filtration and deliquoring data including facility for transient measurements of cake growth and state.

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