Chemotaxis assay using µ-slide Chemotaxis

Transcription

1 Chemotaxis assay using µ-slide Chemotaxis 1. General information The µ-slide Chemotaxis is a tool for observing chemotactical responses of adherent migrating cells over extended periods of time. The linear concentration profile which is required for chemotactical movement is generated by diffusion and stable for at least 48 hours. Three chambers on one slide allow parallel chemotaxis experiments. The following protocol is adapted for adherent cells. chamber configuration: 1 Observation and cultivation area (2000x1000x70 µm) 2 Upper reservoir (40 µl) Lower reservoir (40 µl) 4 Cell inlet with pipet adapter 5 Cell inlet with pipet adapter 6 Pipet adapter 7 Pipet adapter 8 Plugs 9 Cultivation caps Handling movies can be found on 2. Principle Two large volume reservoirs are connected by a thin slit. The reservoirs (2, ) contain different chemoattractant concentrations (indicated below by red and blue color). Inside the connecting slit a linear and stable concentration profile is generated by diffusion. cross section indicating the place of cells, chemoattractant, and gradient Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 1 of 8

2 . Equipment We highly recommend the following equipment to conduct real time chemotaxis experiments with the µ-slide Chemotaxis: adherent cells optimal conditions for cells (heated or incubated stage) inverted microscope (phase contrast, fluorescence, ) motorized stage to observe all chambers in parallel time lapse video equipment (CCD camera, video camera) For all protocol steps we recommend a beveled 200 µl pipet tip (e.g. Greiner ) for filling the pipet adapters right. Please see the sketch for proper pipet usage. 4. Coating µ-slide Chemotaxis recommended 200 µl pipet tip and usage µ-slide Chemotaxis with our hydrophobic uncoated surface (8001) permits no direct cell growth. The surface needs to be coated or the precoated µ-slide Chemotaxis, Collagen IV, sterile (8002) should be used. Please use the following protocol for Collagen IV coating (0.8 µg/cm 2 ): Dilute the Collagen IV (e.g. BD Cat.-Nr. 562) to a concentration of 16 µg/ml using 0.05 M HCl. Fill 80 µl coating solution per chamber using the pipet adapter 7 (step A) until the chamber is completely filled (step B, C, D). Please note that filling the chamber with cells or washing buffer is difficult as long as it is not completely dried. Leave at room temperature for at least 0 minutes. Completely aspirate the solution using a cell culture aspirator. Wash with 80 µl ultra-pure water and aspirate again. Let the chamber dry at room temperature overnight. Work under sterile conditions. Other coatings can be adapted using the same procedure. Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 2 of 8

3 5. Experiment a) Seeding cells The day before seeding the cells and conducting the experiment we recommend placing the cell medium and the µ-slide into the incubator for equilibration. This will prevent the medium inside the slide from emerging air bubbles over the incubation time. Unpack the µ-slide Chemotaxis and put in on a µ-slide rack. Prepare cell suspension as usual. Use cell suspension of approx. x 10 6 cells/ml. High cell concentrations are needed due to the small height of the observation area. Close pipet adapters 6 and 7 by plugs. (not shown) Use a 20 µl pipet (e.g. Gilson P-20) and apply 6 µl cell suspension onto pipet adapter (4). Use the same pipet settings and aspirate air from the opposite pipet adapter (5). Press the pipet tip directly into the adapter. The cell suspension from adapter 4 will be flushed inside filling the entire channel homogeneously. Leave both pipet adapters (4 and 5) filled with cell suspension. Cover pipet adapters 4 and 5 with cultivation caps (no plugs). Control cell density and cell distribution by phase contrast microscopy. Incubate the slide inside a sterile and humid atmosphere to minimize evaporation until cells have attached. This should occur within 1-5 hours depending on your cell type. Decrease evaporation by using Olaf, our humid chamber for cell cultivation. Please visit for Application Note 12 Avoiding evaporation using a humidifying chamber (Olaf). Control cell attachment and morphology by phase contrast microscopy. Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page of 8

4 Optionally, after cell attachment you can remove non-adherent cells by the following steps: Remove both cultivation caps from the pipet adapters 4 and 5. (not shown) Fill 10 µl cell free medium onto pipet adapter 4. Don t trap air bubbles. Aspirate the same amount of liquid from cell inlet adapter 5 as shown. Repeat this step if necessary. b) Filling the reservoirs Remove both cultivation caps from the pipet adapters 4 and 5. Close cell inlet adapter 5 by transferring the plug from adapter 7. Keep in mind that an air bubble might be trapped when closing an empty adapter. Always be sure the adapters are completely filled. Fill the reservoirs by gently injecting medium through cell inlet adapter 4 (step A). Use 40 µl and the recommended pipet tip. In this step it is crucial not to detach cells. This can be achieved by very gently pipetting. Continue until reservoir and the corresponding adapter 7 are completely filled. (step B). The longer the filling takes the fewer cells are stressed. Approx. 0 seconds are fine. Close cell inlet adapter 4 by transferring the plug from adapter 6. (step C) Continue filling by using the pipet adapter 7 (step D). Use 40 µl medium. Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 4 of 8

5 Now, the chamber is completely filled and cells grow in the observation area only. Control your cells under the phase contrast microscope. Keep in mind that pipet adapters 6 and 7 must be completely filled as shown. c) Apply a chemoattractant in one reservoir Apply 18 µl of chemoattractant onto pipet adapter 7 as shown. Don t trap air bubbles. Don t inject the chemoattractant directly (step A). Aspirate the same amount of liquid (18 µl) from the opposite adapter 6 as shown in step B. This will flush the chemoattractant into reservoir. Don t aspirate too fast as this might detach some cells. Close both reservoir adapters (6, 7) with plugs starting with the non-attractant containing side (step C). Don t trap air bubbles. After a short time the chemoattractant will diffuse through the observation area and establish a linear concentration profile (see D) over the cells. See 11. (fluorescence measurements) for details. Please visit for movies on handling µ-slide Chemotaxis! Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 5 of 8

6 6. Calculation chemoattractant concentrations Measurements showed that the maximum working concentration (C 100 ) reaching the cells is only % of the applied concentration (C applied ). That fact is due to the chemoattractant dilution and gradient establishment. Calculation example: Attractant concentration profile over the cells between 100 µg/ml (C 100 ) and 0 µg/ml (C 0 ). Required solutions: Medium with 0 µg/ml for filling the reservoirs (step 6b) Medium with 00 µg/ml for applying into one reservoir (step 6c) Result: Concentration profile over the cells in steady state reaches between 100 µg/ml and 0 µg/ml attractant. 7. Conduct your experiment Mount the µ-slide Chemotaxis on the stage of your inverted microscope and observe cell movement within the observation area. Depending on the cells velocity 1 frame per 1-10 minutes should be sufficient. Depending on your cells requirements heating and incubation devices are necessary. 8. Tracking cells After the experiment we recommend to track the cells with appropriate software. We suggest e.g. Manual Tracking, an ImageJ plugin able to quantify movement of objects between frames of a temporal stack. ImageJ is available here: The Manuel Tracking plug-in by Fabrice Cordelières is available here: 9. Analyzing chemotaxis Phase contrast image with cells paths tracked using Manual Tracking We provide a free ImageJ plug-in for plotting and analyzing the tracked data. Please visit for free download of: `Chemotaxis and Migration Tool`. Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 6 of 8

7 10. Fluorescence measurements Fluorescence measurements with Alexa488 (diffusion coefficient D=16 µm 2 /s) at room temperature (22 C) showed the concentration profile is linear over the 1 mm observation area. The profile is established after 1-8 h. After that it reaches steady state. The profile is maintained linear for over 48 h. After that it slowly flattens due to the concentration equilibration between the reservoirs. 11. Different chemoattractants and time dependencies If you use a chemoattractant with different molecular weight M molecule (and diffusion constant D molecule), different temperatures T, and solutions with different dynamic viscosities η solution, diffusion will be faster or slower. The following example (Alexa488 in water vs. VEGF in endothelial cell growth medium) shows how to compare our fluorescence measurements with different chemoattractants in different solutions. parameters: η = 1 10 η Alexa488 (22 C) EC growth medium (7 C) - kg/s m = kg/s m T 1 = 7 C = 10 K T 2 = 22 C = 295K To simplify matters the diffusing molecules are assumed to be globated. The hydrodynamic radius is therefore dependent on the molecular weight only: R h M molecule with: M VEGF = 45,000 Da M Alexa488 = 570Da Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 7 of 8

8 comparison: D D D D VEGF Alexa488 VEGF Alexa488 k BT1 6 π η1 R = k BT2 6 π η R = 0. 2 h1 h2 = T1 η1 R T2 η R 2 h1 h2 = η η T EC growth medium (7 C) T Alexa 488 solution (22 C) 1 2 M M VEGF Alexa488 = K kg/s m 295 K kg/s m 45,000 Da 570 Da By using VEGF in endothelial growth medium as chemoattractant at 7 C the diffusion is slower with factor. So, the concentration profile is build up slower with that factor. The diffusion of different chemoattractants can be calculated identical. 12. Troubleshooting Air bubbles Emerging air bubbles will disturb the diffusion driven concentration gradient by convection. This should be avoided by equilibrating all media and equipment for an appropriate time (overnight) in the incubator. Avoid unfilled pipet adapters and air bubbles inside the pipet tip! Avoid unfilled pipet adapters and air bubbles inside the pipet tip! Inhomogeneous cell distribution Inhomogeneous cell distribution negatively overlays directed migration and should be avoided. Carefully do all steps following the protocol at No. 6a to avoid flushing cell suspension into the large reservoirs. Temperature instabilities Major changes in temperature during the experiment have may cause convection thus disturbing the diffusion driven gradient. Version 2.0, Prepared by Elias Horn, March 8th, 2007 Page 8 of 8