ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm
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1 Version 1 Last updated 29 June 2018 ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm For the measurement of cell invasion in response to stimuli. This product is for research use only and is not intended for diagnostic use. Copyright 2018 Abcam. All rights reserved
2 Table of Contents 1. Overview 1 2. Materials Supplied and Storage 2 3. Materials Required, Not Supplied 3 4. General guidelines, precautions, and troubleshooting 4 5. Reagent Preparation 5 6. Assay Procedure 6 7. Data Analysis FAQs / Troubleshooting Typical Data Notes 14 Copyright 2018 Abcam. All rights reserved
3 1. Overview Cell Invasion Assay (Basement Membrane), 24-well, 8 µm (ab235882) utilizes a Boyden chamber coated with Basement Membrane Extract (BME), where the cells invade the matrix and then migrate through a semi-permeable membrane in the Boyden chamber in response to stimulants or inhibitory compounds. The percent cell invasion can be analyzed directly in a plate reader. The assay is easy to use, sensitive and adaptable to high-throughput systems. Prepare cells. Prior to the assay, starve cells for hr in serum-free media. Set up cell invasion assay containing desired chemoattractant in the bottom chamber. Incubate the Cell Invasion Chamber at 37 C in CO 2 incubator for 2-48 hrs. Prepare Standard Curve for each cell type. Wash Cells. Add Cell dye and incubate at 37 C in CO 2 incubator for 60 minutes. Disassemble the Cell Invasion Chamber, remove the top chamber and read the bottom well at Em/Ex = 530/590 nm. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 1
4 2. Materials Supplied and Storage Store kit at -20 C in the dark immediately on receipt and check below for storage for individual components. Kit can be stored for 1 year from receipt, if components have not been reconstituted. Components are stable for 6 months after the first thaw. Aliquot components in working volumes before storing at the recommended temperature. Avoid repeated freeze-thaws of reagents. Item Quantity Storage temperatur e (before prep) Storage temperatur e (after prep) Wash Buffer 100 ml -20 C -20 C Cell Dissociation Solution 14 ml -20 C -20 C Control Invasion Inducer 1.5 ml -20 C -20 C Cell Dye 2 x 1 ml -20 C -20 C Cell Invasion Chamber 1-20 C -20 C Basement Membrane Solution 3 x 1 ml -20 C -20 C ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 2
5 3. Materials Required, Not Supplied These materials are not included in the kit, but will be required to successfully perform this assay: Fluorescence Plate Reader. Cotton Swab. Centrifuge to spin 96-well plate. 96-well clear bottom white plate. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 3
6 4. General guidelines, precautions, and troubleshooting Please observe safe laboratory practice and consult the safety datasheet. For general guidelines, precautions, limitations on the use of our assay kits and general assay troubleshooting tips, particularly for first time users, please consult our guide: For typical data produced using the assay, please see the assay kit datasheet on our website. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 4
7 5. Reagent Preparation Briefly centrifuge small vials at low speed prior to opening. 5.1 Wash Buffer 1. Ready to use as supplied. 2. Bring to 37 C before use. 3. Stable for six months after the first thaw. 5.2 Cell Dissociation Solution 1. Ready to use as supplied. 2. Bring to 37 C before use. 3. Stable for six months after the first thaw. 5.3 Control Invasion Inducer 1. Ready to use as supplied. 2. Bring to 37 C before use. 3. Stable for six months after the first thaw. 5.4 Cell Dye 1. Ready to use as supplied. 2. Aliquot and store at -20 C. 3. Bring to 37 C before use. 5.5 Cell Invasion Chamber 1. Open under sterile conditions. 5.6 Basement Membrane Solution 1. Thaw vials as needed slowly on ice or in frost-free 4 C refrigerator. Temperatures above 4 C will rapidly turn the Basement Membrane Solution into a gel. Thawing may take overnight at 4 C. The thawed matrix can be stored at 2-8 C for one week. 2. For long term (6 months) storage, we recommend aliquoting into several tubes according to use and storing at -20 C. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 5
8 6. Assay Procedure Equilibrate all materials and prepared reagents to room temperature just prior to use and gently agitate. Assay all standards, controls and samples in duplicate. 6.1 Cell Invasion Assay Protocol: 1. Add 100 µl of Basement Membrane Solution to coat desired wells of the Top Chamber. Incubate plate at 37 C in an incubator for 1 hour to gel the Basement Membrane Solution. 2. Grow cells of interest in desired media and culture conditions. Grow enough cells to perform a Cell Invasion Assay and a Standard Curve. 3. Adherent cells should be cultured to ~80% confluence. 4. Prior to the assay, starve cells for hours in a serum-free media (0.5% serum can be used, if needed). 5. After starvation, harvest the cells and centrifuge at 1,000 x g, for 5 minutes to pellet cells. 6. Resuspend cell pellet in Wash Buffer and count the number of cells using hemocytometer or automated cell counter. 7. Resuspend cells at 1 x 10 6 cells/ml in a serum-free media. 8. Under sterile conditions, disassemble the Cell Invasion Chamber and carefully remove the plate cover and the top chamber. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 6
9 Figure 1. Cell Invasion plate: The cells are added to the Top Chamber and the Control Invasion Inducer or chemoattractant are added to the Bottom Chamber. 9. Bottom Chamber: Add 600 µl of medium per well containing desired chemoattractant to the bottom chamber. 10. In control well(s), we recommend omitting the chemoattractant. 11. For Positive Control, add 60 µl of Control Invasion Inducer to 540 µl of medium in the bottom chamber. 12. Reassemble the top and bottom chambers while ensuring no air bubbles are trapped between them 13. Top Chamber: Add 200 µl (2-3 x 10 5 cells) of cell suspension to each well of the top chamber. 14. Add desired stimulator or inhibitor to the top well, and gently mix. 15. Carefully replace the plate cover and incubate the Cell Invasion Chamber at 37 C in CO 2 incubator for 2-48 hours. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 7
10 6.2 Standard Curve: 1. Each cell type requires a separate Standard Curve. Prepare a Standard Curve by adding 50 µl cell suspension (1 x 10 6 cells/ml, 50,000 cells) per well in a 96-well plate (white plate clear bottom). 2. Serially dilute the cells 1:1 in Wash Buffer and generate a Standard Curve of cells (50,000, 25,000, 12,500, 6,250, 3,125, 1,562 and 781) in 100 µl total volume. 3. As blank, use 100 µl of Wash Buffer. 4. Add 10 µl of Cell Dye to each well. 5. Incubate at 37 C for 1 hour. 6. Read the fluorescence at Ex/Em = 530/590 nm. 7. Plot the Standard Curve of Number of Cells vs RFU obtained. 8. Fit the data points using a linear trendline with zero intercept. 9. The equation for the straight line and R-square value are used for data analysis of samples. Note: The Cell Invasion RFU reading should fall in the linear range of the Standard Curve. We recommend using triplicates for Standard Curve. 6.3 Separation of Invasive Cells: 1. After the desired incubation with cell invasion inducers/inhibitors, carefully remove the plate cover and aspirate media from the top chamber without puncturing the membrane and matrix. 2. Remove cells from the top chamber using a cotton swab. Disassemble the Cell Invasion Chamber by removing the top chamber. 3. Invert the top chamber and set it aside. Place the plate cover on top of bottom chamber and centrifuge the plate at 1,000 x g for 5 minutes at room temperature. 4. Carefully aspirate the media from the bottom chamber, and wash the chamber with 500 µl Wash Buffer. 5. Centrifuge the plate at 1,000 x g for 5 minutes at room temperature and aspirate the media from the bottom chamber. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 8
11 6.4 Count Invasive Cells: 1. For every twenty wells to be assayed, prepare a mix of 1 ml of Cell Invasion Dye in 10 ml of Cell Dissociation Solution. Mix well. 2. Add 550 µl of the mix to each well of the bottom chamber. 3. Reassemble the Cell Invasion Chamber by placing the top chamber into the bottom chamber. Incubate at 37 C in CO 2 incubator for 60 minutes. 4. After incubation, disassemble the Cell Invasion Chamber, remove the top chamber and transfer 110 µl of mix from the bottom chamber to the 96-well white plate (the same plate having Standards). 5. Read the plate at Ex/Em = 530/590 nm. Multiply the reading by 5 to account for the 5X higher volume in each well of the 24-well plate. Note: During incubation with Cell Dissociation Solution/Cell Invasion Dye, gently tap the plate on the side to ensure optimal dissociation of the invasive cells that cling to the outer side of the top chamber. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 9
12 7. Data Analysis 1. Calculate the number of cells invaded using the equation of the straight line obtained from Standard Curve. 2. Percentage Invasion can be calculated as follows: % Invasion = # Cells in Lower Chamber Total # Cells added to Top Chamber * 100 ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 10
13 8. FAQs / Troubleshooting General troubleshooting points are found at ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 11
14 9. Typical Data Data provided for demonstration purposes only. Figure 2. Standard Curve: HT-1080 cells were harvested, counted and serially diluted to obtain desired cell number. Cells were incubated according to the protocol with Cell Invasion Dye and fluorescence (Em/Ex 530/590) was measured. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 12
15 Figure 3. Cell Invasion: 3T3-NIH and HT-1080 cells were starved overnight and treated with Control Invasion Inducer or remain untreated (No Treatment). Treatment with Control Invasion Inducer demonstrated a significant increase in invasion of HT 1080 cells as compared to 3T3-NIH control cells. ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 13
16 10. Notes ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 14
17 ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 15
18 ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 16
19 ab Cell Invasion Assay (Basement Membrane), 24-well, 8 µm 17
20 Technical Support Copyright Abcam. All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. Austria wissenschaftlicherdienst@abcam.com France supportscientifique@abcam.com Germany wissenschaftlicherdienst@abcam.com Spain soportecientifico@abcam.com Switzerland technical@abcam.com Deutsch: Français: UK, EU and ROW technical@abcam.com +44(0) Canada ca.technical@abcam.com US and Latin America us.technical@abcam.com Asia Pacific hk.technical@abcam.com (852) China cn.technical@abcam.com Japan technical@abcam.co.jp +81-(0) Singapore sg.technical@abcam.com Australia au.technical@abcam.com +61-(0) New Zealand nz.technical@abc.com +64-(0) Copyright 2018 Abcam. All rights reserved
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