-Using Neuro Probe chemotaxis chamber


第二生化マニュアル目次

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 μm). 
The poresize optimal for your type of cell should be confirmed in the 
literature. 
   3 μm: for  PMN
   5 μm: for macrophages, EoL-1, HL-60 
   8 μm: for CHO and THP-1 cells, COS and HEK-293 cells  
   5 μm (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 μl) to eppendorf tube and atore at -80℃.
   In hybridization bag, pour 10 ml of PBS (-) and 100  μl 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 μl. However add about 33 μl 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℃ 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 μl, 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 μl  
   HL-60 cells: 2 x 10E5 cells/100 - 200 μl
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 μM 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.

第二生化マニュアル目次