Plate cells in 96 well plate at 3x103 to 1x105 cells/well. Seeding density depends on purpose and cell line used. For growth curves, cells should be seeded at approx. 3x103 cells/well. For binding or toxicity assay, cells should be seeded at 3x104 to 1x105 cells/well.
Seeding densities can also be based on TC area and split ratio. For growth curves split cells from near confluence at approx. 1:32 to 1:16 (if cells can stand this). For binding and toxicity, seed cells from near confluence approx. at 1:4 to 1:2. These splits should be adjusted if harvested flask is not near confluence or is overconfluent (avoid using overconfluent cells). For calculations the plating area per 96 well plate (center wells) is 60 wells x 0.33 cm2/well = 20 cm3. Mix cell suspension so that cells harvested from 10 cm2 are suspended in each 6 ml of media for a 1:2 split. Cells should be counted to determine and record seeding density.
To plate cells, harvest flask of cells, transfer to sterile tube, and dilute in plating media to approx. 106 cells/ml (estimated). Mix well and remove aliquot for counting (place tube of cells on ice while counting aliquot).
Count with hemocytometer of Coulter counter.
Note: For cell growth curves, flasks of cells should be harvested at subconfluence. For special purposes cells may be serum starved or GLN starved for 18 to 24 hours prior to harvesting for plating. Cells at high density should never be used because clumps of cells will result.
Prepare 96 well plates to be seeded: open packages, label, pre-dose, etc. For example, for dose response curves, drugs can be pre-diluted in plates leaving 100l/ well of 2X drug concentrations. Addition of 100 l/well of cell solution will result in final 1X drug concentration.
Dilute cells to 1x104 to 3x105 cells/ml in plating media. Mix >6 ml of dilution per plate to be seeded. Mix well and transfer diluted media to sterile multipipetter trough.
Load multipipetter with 6 or 10 tips depending on seeding strategy: by column (6) or by row (10). Plate seeding can affect results if one barrel of multipipetter consistently delivers more or less volume of cell suspension. In general one should seed by rows if test groupings will be by columns.
Using multipipetter, carefully pipette 100 l/well of diluted cell suspension into CENTER 60 WELLS of 96 well plate (10x6 matrix). When drawing up cell suspension, expel once to mix suspension in trough. After drawing up 100 l/tip, visually confirm that liquid levels are equal in each tip.
If drugs, etc. were not pre-added to wells, add 100 ml/well of media containing 2X concentration of substance to each column of wells. This can be done immediately or after the cells have adhered overnight. Waiting will avoid drug effects on seeding efficiency. For growth curves, add drugs on same day cells are seeded.
Fill exterior wells with 200 to 250 l/well of sterile H2O or PBS using repeater pipette. This will reduce evaporation from inner wells.
Incubate plates.
Analyze plates at various times after seeding according to purpose. For growth curves, times will depend on growth rate of cells. For fast growing cells, plates should be analyzed at 1, 2, 3, 4, 5 and 7 days. For intermediate and slow growing cells, plates should be analyzed at 1, 3, 5, 6, 10, and 14 days with a media change at day 7.
MTT ASSAY: to determine relative cell density.
Prepare MTT solutions.
10X MTT: Completely dissolve 1 tablet phosphate buffered saline pH 7.4 in 200 ml MQ water. In a 200 ml beaker, add 100 ml of this to 0.5 g MTT. Cover and stir 4 -24 hours at 4oC. DO NOT HEAT. Filter through 0.2mm filter into a sterile 100 ml bottle. Store at 4oC.
HCl in Isopropanol: Add 1 ml 12 N HCl to 299 ml isopropanol in a 500 ml bottle. Store at 4oC.
Dilute 1 part 10X MTT solution with 9 parts serum free media (media without FBS is okay). May want to use media without phenol red.
Remove media from wells by dumping off and blotting upon paper towels. Plates must be shaken hard when dumping due to surface tension in wells. Give the plates several hits on the towels to ensure all media is removed. Check through microscope to ensure cells were not lost during media removal.
Fill all wells with 100 µl/well serum-free media plus MTT.
Incubate plate for 2 to 4 hours (4 hrs probably better). Must be consistent if comparing results from different plates.
Add 100 µl/well of acidic isopropanol solution.
Aggressively triturate solution in wells using micropipetter, to detach cells and break up purple crystals. Avoid bubbles and spillage.
Place plate on rocker for 30 minutes.
Read optical densities on plate reader using 560 nm filter with 650 nm reference filter. OD560-650 should be proportional to cell number. Columns or rows of outer wells originally filled with PBS should be blank.