Chemotaxis assay (Ingvar Ferby, Takashi Izumi)

-Using Neuro Probe chemotaxis chamber


‘æ“ñ¶‰»ƒ}ƒjƒ…ƒAƒ‹–ÚŽŸ Material and equipment: i) Neuro Probe 96-well chemotaxis chamber (model: AB96 ) ii) Framed polycarbonate filter (PVP-free). Pore sizes available in our lab ( 3, 5, 8 ƒÊm). The poresize optimal for your type of cell should be confirmed in the literature. 3 ƒÊm: for PMN 5 ƒÊm: for macrophages, EoL-1, HL-60 8 ƒÊm: for CHO and THP-1 cells, COS and HEK-293 cells 5 ƒÊm (PVP+: standard) : eosinophils iii) "Diff Quik" staining set, or equivalent. Fibronectin (FN) coating of the filter Wako: 300-00571 (1 mg) or 300-00573 (5 mg) Add 1 or 5 ml H2O to obtain 1mg/ml solution. Devide an aliquot (100 ƒÊl) to eppendorf tube and atore at -80Ž. In hybridization bag, pour 10 ml of PBS (-) and 100 ƒÊl FN, and soak the filter. Shake it for 30 min. Wipe the extra solution with KIM-WIPE, and air-dry the filter. Chamber preparation: 1) Unscrew and separate the lower compartment from the upper. Make sure the compartments are clean and dry. 2) Orient the bottom plate so that the NP trademark is at the upper left. Fill the lower wells with chemoattractant or control medium wich has been warmed to 37degree and vortexed in order to remove dissolved gas. The wells take a volume of 30 ƒÊl. However add about 33 ƒÊl so that a small positive meniscus will form. This will prevent airbubbles from getting trapped when the filter makes contact. 3) The framed filter should be snapped in place between the iron taps of the upper plate, so that the frame makes close contact with a maximal number of taps. The blank side of the filter must face the upper wells. 4) With the filter installed, invert the top plate onto the filled bottom plate, carefully and without rocking. 5) Tighten all thumb nuts gradually and equally. Preincubate the chamber for 37Ž for 30 min. Preparation and addition of responding cells: 6) Cells are prepared and suspended in a suitable medium (suggestively serum free). The upper well volume is 225 ƒÊl, even though the volume of the cellsuspention to be added is not critical. The optimal number of cells to be added to one well vary greatly (10 000 - 1 000 000) depending on 1) kind of cells, 2) their size and behavior, 3) ligand used etc. and will have to be tested. However it seems like if only a big number of cells make a final spectrophotometrical detection of stained cells possible. (The alternative is timeconsuming cell counting) CHO cells: 1 x 10E5 cells/100 - 200 ƒÊl HL-60 cells: 2 x 10E5 cells/100 - 200 ƒÊl 7) To measure chemokinesis (undirectional cellmobility), add a small volume of chemoattractant to the upper wells so that it's final concentration correspond to that in the lower compartment. 8) Incubate chamber at 37degree, 5% CO2. The optimal incubation time vary depending on celltype, ligand etc. (ex.: PMN 1-2 hrs, macrofage 3-5 hrs, CHO cells 3-4 hrs). Removal and staining of filter: 9) Remove fluid from the upper well by shaking the chamber up side down over a sink. 10) Remove thumb nuts and free the filter. 11) Fix the filter ~30 sec (3 min) in methanol. Then stain for ~30 sec (3 min) in "Diff Quik" solution I, followed by ~30 sec (3 min) in solution II. Rinse in distilled water. Wipe of the nonmigrated cells on the blank side of the filter with Kleenex or equivalent. Rinse again in water for a couple of minutes. Set the filter aside to dry up. 12) The filter can be read on the Bio-Rad plate reader at 595 nm. In case the staining is undetectableby plate reader, mark the areas representing each well, mount the filter on glycerol and count the cells attached to the filter. Warnings: a) After usage, quickly wash the chamber components in water/ 1 drop of detergent (mama-lemon) and wash it with a soft sponge and rince it with water and distilled-water. Dry it in air. Lipids and protein tend to accumulate on the chamber walls and might contaminate later assays. b) The equipment must not be exposed to high tempratures, autoclaved or washed in dish machine. c) Be careful not to put your fingers on the filter before use, sinse they are full of possible chemoattractants. d) If the timeintervall between completed chamber preparation (step 5) and addintion of cellsuspention (step 6) is made to long, water will evaporate through the filter and pull air into the lower compartment. Option: Instead of Diff Quik, one can use fluorimager with stained cells. Cells are stained with Caicein-AM (Molecular Probes, C-3099 1 ml, 1 mg/ml DMSO = 1 mM). 4 ƒÊM for 30 min in any medium (with FCS or BSA is OK). Wash cells twice using cfg (3000 rpm for 3 min in Tomy MC-150). Resuspend in chemotaxis medium. After incubation, wipe of the nonmigrated cells on the blank side of the filter with Kleenex or equivalent. Soak kleenex in PBS(-) and wipe twice the nonmigrated side. Dry the filter with dry Kleenex. Count the fluorescense of the filter with fluoroimager (FluorImager595, Molecular Dynamics). Emission: 488 nm, Filter: 530DF30, PMT voltage: 700 V. ‘æ“ñ¶‰»ƒ}ƒjƒ…ƒAƒ‹–ÚŽŸ