PROTOCOL 1850 Millrace Drive, Suite 3A Eugene, Oregn 97403 In-Cell ELISA Supprt Pack Adherent Cell Prtcl MS921 DESCRIPTION This In-Cell ELISA (ICE) Supprt Pack prtcl is fr use with adherent cells. It cntains enugh materials t perfrm five 96 well micrplate ICE experiments with MitSciences ICE-validated antibdies. A cell culture micrplate is nt prvided in this pack, as an alternative MitSciences ffers a supprt pack cntaining cell culture micrplates (MS922). Fr an ICE assay it is necessary t purchase bth primary antibdy(ies) and labeled secndary antibdy(ies). Antibdies are sld separately, allwing custmizatin f the target(s) f interest, methd f detectin and multiplexing. Fr IR detectin a LI-COR system is necessary. Fr HRP detectin HRP substrate slutin and a standard micrplate reader are required. Fr nn adherent cells r cells likely t detach see ur suspensin cell prtcl MS922. Rev.1 Kit Cntents: Strage: Part Number Item Amunt 8209725 10X Phsphate Buffered Saline (PBS) 250 ml 8209726 100X Tritn X-100 (10% slutin) 1.25 ml 8209727 400X Tween-20 (20% slutin) 4 ml 8209728 10X Blcking Slutin 40 ml 8209729 1X Janus Green Stain 30 ml Stre all cmpnents upright at 4 C. PRINCIPLE In-Cell ELISA Adherent Cells uses quantitative immuncytchemistry t measure prtein levels r psttranslatinal mdificatins f cultured adherent cells. The cells are grwn in and fixed t the bttm f a cated 96-well plate (prvided). Targets f interest are detected by primary antibdies, which are in turn quantified with labeled secndary antibdy(ies). MitSciences ffers highly-specific, well-characterized primary antibdies, IRDye - r HRP-labeled anti-muse and anti-rabbit secndary antibdies, as well as IRDye -labeled istype specific anti-muse antibdies. By cmbining antibdies f different species r istype and apprpriate IR-labeled secndary antibdies, tw clr multiplexing can be achieved in the 800/700 channels. IR imaging and quantificatin is perfrmed using a LI-COR Odyssey r Aerius system. HRP-labeled cmplexes are develped and quantified clrimerically using spectrphtmeter. LI-COR, Odyssey, Aerius, IRDye and In-Cell Western are registered trademarks r trademarks f LI-COR Bisciences Inc. BACKGROUND This prtcl is a general prtcl fr ICE analysis and can be used with any f MitSciences' ICEvalidated antibdies. Specific scientific infrmatin, backgrund and wrking cncentratin fr each antibdy are detailed in each antibdy s crrespnding Technical Data Sheet.
In-Cell ELISA Adherent Cells Prtcl ADDITIONAL MATERIALS REQUIRED Apprpriate cell culture micrplates with a surface cating t allw cell attachment. Cell culture treated plates must have clear bttm wells and black walls are recmmended (cnsult MitSciences fr advice) MitSciences ICE-validated primary antibdy(ies) Apprpriate MitSciences 1000X IRDye - r HRP- labeled secndary antibdy(ies) Fr HRP detectin HRP slutin substrate is necessary such as KPL Sureblue TMB slutin. Adequate instrumentatin. Fr IRDye use a LI-COR Odyssey r Aerius near-infrared imaging system. Fr HRP detectin use a spectrphtmeter plate reader (Mlecular Dynamics SpectraMax recmmended) capable f measuring absrbance at 650 (preferably in a kinetic mde) r 450 nm. 20% parafrmaldehyde (Electrn Micrscpy Sciences #15713) Deinized water Multichannel pipette (recmmended) 0.5 M HCl (ptinal fr Janus Green cell staining prcedure) ASSAY PROTOCOL Nte: Be cmpletely familiar with the prtcl befre beginning the assay. D nt deviate frm the specified prtcl steps r ptimal results may nt be btained. A. Preparatin f sufficient buffers and wrking slutins t analyze a single micrplate 1. Prepare 1X PBS by diluting 45 ml f 10X PBS in 405 ml Nanpure water r equivalent. Mix well. Stre at rm temperature. 2. Prepare 1X Wash Buffer by diluting 0.625 ml f 400X Tween-20 in 250 ml f 1X PBS. Mix well. Stre at rm temperature. 3. Immediately prir t use prepare 8% Parafrmaldehyde Slutin by mixing 6.25 ml Nanpure water, 1.25 ml10x PBS and 5.0 ml 20% parafrmaldehyde. Nte Parafrmaldehyde is txic and shuld be prepared and used in a fume hd. Dispse f parafrmaldehyde accrding t lcal regulatins. 4. Immediately prir t use prepare 1X Permeabilizatin Buffer by diluting 0.25 ml 100X Tritn X- 100 in 24.75 ml 1X PBS. Mix well. 5. Immediately prir t use prepare 2X Blcking Slutin by diluting 5 ml 10X Blcking Slutin in 20 ml 1X PBS. 6. Immediately prir t use prepare 1X Incubatin Buffer by diluting 2.5 ml 10X Blcking Slutin in 22.5 ml 1X PBS. Page 2 f 8
In-Cell ELISA Adherent Cells Prtcl B. Cell Seeding Nte A blank plate map fr ntatin f treatments and cells seeding is prvided n page 8. Adherent Cells: 1. Adherent cells can be grwn in the Assay Plate r seeded directly int the Assay Plate and allwed t attach fr several hurs t vernight. The ptimal cell seeding density is cell type dependent. Fr suggestins regarding the cell seeding see Appendix. As an example, HeLa cells shuld be seeded between 25,000 and 50,000 cells per well. 2. The attached cells can be treated as desired. Fr suggestins regarding the treatment t induce apptsis, psitive and negative cntrls see Appendix. Nte When treatment with drug f interest is perfrmed, it is recmmended t include wells with untreated cells and cells treated with the vehicle nly. 3. After treatment prceed t Step C1. C. Cell Fixatin 1. Immediately add an equal vlume (100 µl) f 8 % Parafrmaldehyde Slutin t the wells f the plate cntaining culture media. 2. Incubate fr additinal 15 minutes. 3. Gently aspirate the Parafrmaldehyde Slutin frm the plate and wash the plate 3 times briefly with 1X PBS. Fr each wash, rinse each well f the plate with 300 µl f 1X PBS. Finally, add 100 µl f 1X PBS t the wells f the plate. The plate can nw be stred at 4 C fr several days. Cver the plate with prvided seal. Fr prlnged strage supplement PBS with 0.02% sdium azide. Nte The plate shuld nt be allwed t dry at any pint during r befre the assay. Bth parafrmaldehyde and sdium azide are txic, handle with care and dispse f accrding t lcal regulatins. D. Assay Prcedure Nte It is recmmended t use a plate shaker (~300 rpm) during incubatin steps. Any step invlving remval f buffer r slutin shuld be fllwed by bltting the plate gently upside dwn n a paper twel. 1. Remve PBS and add 200 µl f freshly prepared 1X Permeabilizatin Buffer t each well f the plate. Incubate 30 minutes. 2. Remve 1X Permeabilizatin Buffer and add 200 µl f 2X Blcking Slutin t each well f the plate. Incubate 2 hurs. 3. Prepare 1X Primary Antibdy Slutin by diluting MitSciences stck antibdy(ies) int 1X Incubatin Buffer. See Appendix fr mre infrmatin. Page 3 f 8
In-Cell ELISA Adherent Cells Prtcl 4. Remve 2X Blcking Slutin and add 100 µl diluted Primary Antibdy Slutin t each well f the plate. Incubate vernight at 4 C. Nte T determine the backgrund signal it is essential t mit primary antibdy frm at least ne well cntaining cells fr each experimental cnditin and detectr antibdy used. 5. Remve Primary Antibdy Slutin and wash the plate 3 times briefly with 1X Wash Buffer. Fr each wash, rinse each well f the plate with 250 µl f 1X Wash Buffer. D nt remve the last wash until step 8. 6. Prepare 1X Secndary Antibdy Slutin by diluting 12 µl f 1000X labeled-secndary antibdy(ies) int 12 ml 1X Incubatin Buffer. Nte Prtect flurescently labeled antibdies frm light. 7. Remve the 1X Wash Buffer and add 100 µl 1X Secndary Antibdy Slutin t each well f the plate. Incubate 2 hurs. 8. Remve Secndary Antibdy Slutin and wash 4 times briefly with 1X Wash Buffer. Fr each wash, rinse each well f the plate with 250 µl f 1X Wash Buffer. D nt remve the last wash. 9. Based n the chsen MitSciences labeled secndary antibdy(ies), use apprpriate detectin/imaging and instrumentatin. Refer t the prtcls accmpanying the secndary antibdies. Page 4 f 8
In-Cell ELISA Adherent Cells Prtcl E. Whle Cell Staining with Janus Green (Optinal) Nte The (IR r HRP) signal f antibdy-specific cmplexes can be nrmalized t the Janus Green staining intensity t accunt fr differences in cell seeding density. It is recmmended t use a plate shaker (~300 rpm) during incubatin steps. 1. Remve last 1X Wash Buffer and add 50 µl f 1X Janus Green Stain per well. Incubate plate fr 5 minutes at rm temperature. 2. Remve dye, wash plate 5 times in deinized water r until excess dye is remved. 3. Remve last water wash, blt t dry, add 200 µl f 0.5 M HCl and incubate fr 10 minutes. 4. Measure using a LI-COR Odyssey scanner in the 700 nm channel r measure OD595 nm in a standard micrplate spectrphtmeter. F. Data Analysis Nte Analyze data using LI-COR ICW sftware, r ther suitable data analysis sftware, such as Micrsft Excel r GraphPad Prism. 1. Crrect the raw ICE signal fr the backgrund signal by subtracting the mean signal f well(s) incubated in the absence f the Primary Antibdy frm all ther readings. 2. This step is ptinal. Crrect the Janus Green fr the backgrund signal by subtracting the mean Janus Green signal f well that d nt cntain cells frm all ther Janus Green readings. 3. Nrmalize the ICE signal. Divide the backgrund-crrected ICE signal by the (backgrundcrrected) Janus Green signal. Page 5 f 8
In-Cell ELISA Adherent Cells Prtcl FLOW CHART Fr quick reference nly. Be cmpletely familiar with previus details f this dcument befre perfrming the assay. Adherent Cell Seeding Seed cells int Assay Plate. Allw cells t adhere. Treat cells as desired in ttal vlume f 100 µl. Fix Cells (30 minutes) Add an equal vlume (100 µl) f 8% Parafrmaldehyde Slutin t fix and crsslink the cells t the plate. Incubate fr additinal 15 minutes. Wash wells with PBS (may be stred at 4 C at this pint). Primary Antibdy Incubatin (Overnight) Dilute the primary antibdy stck 200X in 1X Incubatin Buffer and add the diluted primary antibdies. Incubate vernight at 4 C. Wash thrughly. Secndary Antibdy Incubatin (2 hurs) Dilute the secndary antibdy stck 1000X in 1X Incubatin Buffer and add the diluted secndary antibdies. Incubate 2 hurs at RT. Wash thrughly. (Develp and) Read Plate As apprpriate, image the plate with an IR scanner r develp the HRP labeled plate and read it with a spectrphtmeter. Exprt data. If desired, stain with Janus Green and measure relative cell seeding density in an IR imager r micrplate spectrphtmeter. Exprt data. Determine backgrund crrected signal and then rati signal t Janus Green if desired. Page 6 f 8
In-Cell ELISA Adherent Cells Prtcl APPENDIX Cell seeding density, culture medium and ther grwth cnditins are imprtant and cell-type specific parameters shuld be defined by the user. Adherent cells can be grwn and treated directly in the Assay Plate. The cating f the Assay Plate des nt interfere with the cell grwth and enhances the cell attachment. Cell attachment can be checked with a micrscpe. When the cells are fully attached the media can be remved and replaced with media cntaining drug f interest. Culture media cntaining up t 10% fetal serum des nt interfere with the cell fixatin and crsslinking t the plate. The cell seeding density f the Assay Plate is cell type dependent. It depends n the cell size (diameter, in case f the adherent cells) and the abundance f the target prtein. As a general guideline, if final fixed cells frm a mnlayer, ICE assays using Mitsciences ICE-validated antibdies give rbust signals. The cell seeding can be determined experimentally by micrscpic bservatin f cell density f serially diluted cells. Fr adherent cells, prepare a serial dilutin f the cells in a plate (f similar well dimensins) and bserve the cell density in a micrscpe. Wrking n the high end f cell densities will generate strnger signals and allw small signal increases t be measured accurately. As an example, HeLa and HepG2 cells shuld be seeded frm 25,000 t 50,000 cells per well, human fibrblasts (HdFN) frm 15,000 t 25,000 cells per well, Wrking n the high end f this range will generate strnger signals and allw greater reductins t be measured accurately, this is imprtant in particular fr less abundant targets r lwer affinity antibdies. It is essential t mit primary antibdy in at least ne well t prvide a backgrund signal fr the experiment which can be subtracted frm all measured data. This shuld be dne fr each experimental cnditin and detectr antibdy used. Primary antibdies are supplied by MitSciences with a recmmended final cncentratin fr ICE which can be fund in the Technical Data Sheet fr each antibdy. The assay can be als perfrmed in 384-well plate frmat. Cntact MitSciences representative t inquire abut 384 well Assay Plates. COMPLEMENTARY PRODUCTS Mitsciences ffers an equivalent In Cell ELISA supprt pack fr Suspensin cells. Mitsciences als ffers a range f ICE-validated primary antibdies. http:///in-cell-elisa.html Mitsciences ffers IR dye cnjugated secndary antibdies fr detectin f muse antibdies and rabbit antibdies as well as istype specific anti-muse antibdies in the 700 and 800 IR channels. By cmbining antibdies f different species r istype and apprpriate IR labeled secndary antibdies, tw clr multiplexing can be achieved in the 800/700 channels. Alternatively Mitsciences als ffers HRPcnjugated secndary anti-rabbit and anti-muse secndary antibdies fr clrimetric detectin. These prducts als include the develpment slutin t cmplete the assay. OTHER RESOURCES MitSciences has published an applicatin guide which prvides an verview f the ICE platfrm and ICE cmpatible antibdies with experimental data frm varius cell treatments. Page 7 f 8
In-Cell ELISA Adherent Cells Prtcl A B C D E F G H / / LI-COR, Odyssey, Aerius and IRDye are registered trademarks r trademarks f LI-COR Bisciences Inc. Page 8 f 8