Motility User Manual CYTOOchip

Transcription

CYTOOchips™ Motility
User Manual
For Research Use only
Refrigerate at 4°C on arrival. Keep bag sealed.
Expiry date is indicated on the bag.
CYTOOchip
A CYTOOchip is a 19.5 x 19.5 mm2, 170 µm thick gridded coverslip of optical grade glass covered with an
organized grid of a 168 individual blocks. Grid coordinates are printed on the underside of the chip (Grid
Positioning System) and enable the user to return to the exact same area over time. The adhesive
micropatterns are printed on the top face of the chip. Each square block, 1.3 x 1.3 mm 2, is composed of an
array of identical patterns.
Grid coordinates start from the upper left corner. Columns are numbered from 1 to 13 and rows from A to M.
Only the grid numbers and arrows are seen under the microscope, the patterns might be visualized only if
fluorescent protein coating is used.
1
Chip identification letter
Allows error-proof CYTOOchip identification
within experiment one (unique every 18 CYTOOchips)
Low fluorescence optical grade glass
Standard coverslip thickness 175%m (1.5)
Cell/ micropattern localization grid
Allows easy cell retrieval over time within 168 blocks
(Block period 1.3mm)
A
2
3
4 5
6
7 8
9
1
0
B
C
D
E
F
G
H
I
J
K
CYTOO logo
Allows easy error-proof chip orientation
(the micropatterns are on the top surface)
L
M
CYTOOchips Motility User Manual PRO-Ext-11 v3
Last modified: 01/06/2014 © CYTOO
1 1
1 2
1
3
Fig. 2. General layout of CYTOOchips with alphanumeric block grid. The block coordinates are printed locally
between the blocks and accompanied by arrows. The block E5 is zoomed as an example. The letters A to M and
numbers 1 to 13 on the periphery are not present on the chip and are given here only for illustration.
CYTOOchip Motility micropatterns
The CYTOOchip Motility has been designed to offer adhesive lines of different widths for 1D cell migration and
large squares or discs for 2D migration (Fig. 2). The chip features the lines of 4 different widths: 2.5, 5, 10 and
20 µm with the line-to-line pitch (period) of 75 µm for all line widths (Table. 1)
1D
2D
Gap
Widt
h
Pitch
Fig. 2. CYTOOchip Motility offers both the tracks for 1D cell migration and the areas for conventional 2D
migration.
Pattern
Width, µm
Gap, µm
Pitch, µm
Lines per block
height (1.3 mm)
Lines
per chip
Line 2.5
2.5
72.5
75
16
192
Line 5
5
70
75
16
192
Line 10
10
65
75
16
192
Line 20
20
55
75
16
192
2D migration
areas
See below for shape and organization of 2D areas
Table 1. Parameters of the micropatterns present on the CYTOOchip Motility.
Layout of the CYTOOchip Motility
The lines are organized over the chip in 4 identical quadrants (Fig.3). Each quadrant is again divided into 4
zones and each line width is arrayed and uninterrupted over 3x3 blocks (line length = 3800µm). There are in
total 192 lines of each width per chip.
The blocks separating the 4 quadrants hold the large 2D migration areas and the discs of 500 µm. See the next
page for details.
Fig. 3. Layout of CYTOOchip Motility with a zoomed region of one of 4 identical quadrants showing 4 adjacent
blocks with 4 different line widths. Note that block M13 is occupied by the CYTOO logo.
2D migration areas
In column 7 and row G, we have placed large 2D migration areas (Fig. 4): squares or rectangles covering the
area of a full block and discs of 500 µm in diameter (4 discs per block). As an added feature, in B7, E7, I7 and
L7 blocks , lines of the neighboring blocks run into the 2D rectangular areas, thus enabling analysis of transitions
from 1D to 2D. 2D migration areas are accessible only using CYTOOchamber™ 1 well (see below).
Fig. 4. CYTOOchip Motility with a zoom on 2D area region. The gap between the discs and 2D areas is 150 µm.
Features
Blocks
2D areas
G2, G5, G7, G9, G12
Discs 500 µm diameter
G1, G3-G4, G6, G8, G10-G11, G13; A7, C7-D7, F7, H7, J7-K7, M7
1D/2D transitions
B7, E7, I7, L7
Table 2. Blocks holding different 2D migration areas.
Compatibility with CYTOOchambers™
The layout of CYTOOchips Motility is compatible both with the 1well and 4well CYTOOchambers for live cell
imaging (Fig. 5). The 4 well CYTOOchamber allows you to carry out time-lapse experiments in 4 different
conditions in parallel with a single chip. Depending on the chamber used some lines at the periphery of the
wells might be covered by the gasket.
Fig. 5. The blue dotted lines give the outline of the wells in the 1 well (left) and 4 well (right) CYTOOchamber
configurations. Note that in 4 well configuration 2D areas are not accessible.
Packaging of CYTOOchips
CYTOOchips are packaged in an embossed clear plastic 6-blister tray sealed
with paper (Fig. 6). Each blister can be opened individually.
The blister is sealed in an aluminum bag under protective atmosphere.
Storage
Refrigerate at 4°C on arrival. Expiry date is indicated on the blister and on
the bag. The blister is sealed in a bag with silica gel to keep the CYTOOchips
under dry conditions. Keep the bag sealed and stored at 4°C. Once opened,
use all chips rapidly.
Fig. 6. CYTOOchips packaging
Instructions for use
For Research Use only
THIS PROTOCOL IS OPTIMIZED FOR HELA AND RPE1 CELLS. TIMES FOR ATTACHMENT AND SPREADING MAY
VARY CONSIDERABLY DEPENDING ON YOUR CELL TYPE.
For time lapse experiments, we highly recommend the use of a CYTOOchamber (available with 1 or 4 wells)
1. CYTOOchips are packaged individually. Remove the paper seal and mount the CYTOOchip in a
CYTOOchamber. (Alternatively you can use a 35 mm culture dish or a 6 well plate but resolution, focus and
imaging will be far from optimal.) Adapt seeding densities appropriately.
- Make sure the silicone gasket is correctly fitted on the main upper body of the CYTOOchamber.
- Place a CYTOOchip in the bottom frame. The “CYTOO” logo should be facing up and cells will be seeded
on this side of the chip.
- Grasp the thin bottom plate carrying the CYTOOchip by
the sides in one hand and slowly bring it into contact
with the main body (Fig. 7). The main body will
automatically attach to the bottom plate by magnetic
force.
2. Prepare your cell suspension and check that cells are well
dispersed. Count cells and adjust the suspension to a density
of 50,000 cells per ml. (This density is simply an indication
and the optimum seeding density may vary considerably
depending on the type of experiment you plan to carry out, Fig. 7. Assembling of the CYTOOchamber
as well as its duration).
3. Dispense 450 µl in the 1well CYTOOchamber (or 100 µl in each of the 4 well CYTOOchamber). Check that
the liquid completely covers the bottom of each well.
4. Cover with the transparent glass lid.
DO NOT ROCK OR SWIRL THE CYTOOCHAMBER AS THIS WILL LEAD TO HETEROGENEOUS SEEDING
DENSITIES OVER THE CHIP
5. Let the cells sediment for 10 min under the hood then move them to the cell incubator. The adhesion
process will start immediately.
6. After 10-30 min, depending on the cell type, cells will start attaching.
7. When cells start spreading, gently fill the CYTOOchamber with 1ml (or 200µl in a 4 well) culture medium.
8. OPTIONAL: Wash gently the surface of the coverslip with 1ml (or 200µl) cell medium a few times to remove
unattached cells.
TAKE EXTREME CARE NOT TO DRY OUT THE SURFACE OF THE CHIP DURING WASHING.
This step will remove all cells that have sedimented onto non-adhesive surfaces and avoid that additional
floating cells roll into the lines during your experiment.
9. After the final wash, top up the CYTOOchamber with cell medium to reach a total of 2ml (or 300µl for the
4well). Place the dish/plate in the cell incubator for at least one hour to allow cells to achieve full spreading.
10. When ready for videomicroscopy, if necessary top up again the wells completely to obtain a plane surface
and reduce meniscus-induced deformations under phase contrast.
11. Place on the 35mm microscope stage adaptor, taking care to orient the chamber so that “CYTOO” is visible
in the right bottom corner. This will orient the grid coordinates and make it easy to find your way around
the CYTOOchip layout.
12. At the end of the live cell observation, cells can be fixed and stained directly in the chamber and then
removed for final mounting. The Grid Positioning System on the CYTOOchip allows easy relocation of the
previously imaged cells for further analysis in immunofluorescence.
Troubleshooting
Observations
Possible solutions
Cells are not homogeneously seeded
over the surface of the chip. There
are more cells in the center of the
chip and very few on the edges.
In Steps 4 and 5, avoid any movement of the CYTOOchamber such
as rocking or swirling.
In Step 5, be sure to keep cells under the hood for 10 min before
moving them to the incubator. Increase this time if cells take a long
time to attach.
Cells do not spread out on the lines.
They keep a round shape and are
bright in phase contrast.
Cells make blebs.
The adhesion protein used to make the lines might not be the
appropriate protein for the adhesion of this cell type. Contact us.
We can offer micropatterns without adhesive proteins that can be
used as such, or with a different adhesive protein.
Technical support
support@cytoo.com. Please use this email address for any questions or requests for information concerning
this product. We will then put you in direct contact with one of our experts.
Ordering information
Cat. No.
Product
Description
10-031-00-06
CYTOOchips Motility A
10-031-10-06
CYTOOchips Motility FN
10-031-13-06
CYTOOchips Motility
FN650
CYTOOchamber 1 well
CYTOOchamber 4 wells
Glass 19.5x19.5 mm; 170 µm; Gridded; Lines: Length 3800 µm, Width
2,5; 5, 10, 20 µm; Discs 500 µm; 2D areas; Activated; Set of 6
2,5; 5, 10, 20 µm; Discs 500 µm; 2D areas; Fibronectin; Set of 6
2,5; 5, 10, 20 µm; Discs 500 µm; 2D areas; FN Fluo exc. 650nm; Set of 6
Round, ext. diam. 35 mm; max. imag. area 16 x 16 mm2; with glass lid
Round, ext. diam. 35 mm; max. imag. area 7.5 x 7.5 mm2 per well; with
glass lid
Chamber with open or closed customized configuration
With clips; accepts up to 6 CYTOOchambers 30-01x
30-010
30-011
30-090
30-020
CYTOOchamber Custom
Microscope adaptor
plate
For inquiries please contact us online www.cytoo.com/contact-us or email sales@cytoo.com.

Motility User Manual CYTOOchip

Transcription

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