[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"doc-detail-165122-en":3,"doc-seo-165122-105":29,"detail-sidebar-cat-1-en-105":89},{"code":4,"msg":5,"data":6},0,"success",{"doc_id":7,"user_id":8,"nickname":9,"user_avatar":10,"doc_module":11,"category_id":12,"category_name":13,"doc_title":14,"doc_description":15,"doc_content":16,"file_id":17,"file_url":18,"file_type":19,"file_size":20,"view_count":4,"is_deleted":4,"is_public":11,"is_downloadable":11,"audit_status":11,"page_count":21,"language":22,"language_code":23,"site_id":24,"html_lang":23,"table_of_contents":25,"faqs":26,"seo_title":27,"seo_description":15,"update_tm":28,"read_time":11},165122,13056703019662,"Evangeline","https://ap-avatar.wpscdn.com/avatar/be000253a8e92610077?_k=1778726343310543188",1,17,"Forms","Antibody Staining in 96-Well Plates - 2017","Protocol for staining surface antigens on cells using a 96-well plate, focused on mononuclear cells derived from peripheral blood. Covers key workflow elements including blocking prior to staining, antibody incubation at room temperature (or on ice with extended time and cold centrifugation), careful washing to avoid cross-well contamination, and fixation with formaldehyde-containing buffer. Includes reagent composition, recommended cell concentration range, centrifugation and wash volumes, and guidance for dark, cold storage and tandem dye limitations.","Antibody staining of surface antigens on cells using a 96 well plate\nNotes\nThis is a general protocol based on staining mononuclear cells from peripheral blood.\nIt does not concern specific antibodies, however, it takes into consideration: Incubation, washing and fixation.\nFor other cell types you might need to optimize the procedure.\nWe recommend blocking before staining.\nFor more details, read Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages by Andersen et al 2016, \u0013 HYPERLINK \"https://www.ncbi.nlm.nih.gov/pubmed/27731950\" \\o \"Cytometry. Part A : the journal of the International Society for Analytical Cytology.\" \u0014Cytometry A.\u0015 2016 Nov;89(11):1001-1009\nIn the following protocol the antibody incubation is performed at room temperature (RT). If you want to incubate on ice the incubation time should be expanded to 30-60 min and the centrifuge should be cold.\nIn each well there will be approximately 15µl left after pouring off your supernatant from a round bottom plate.\nMATERIAL\nStain buffer: PBS pH 7.4 with 0.5% BSA and 0.09% Na-azide\nBlocking Reagent: Human Ig\nFluorocrome conjugated antibodies.\nCells at 1-5x106/ml in stain buffer\nA round bottom or V bottom microwell plate\nFixation buffer: PBS pH 7.4 with 0.9% formaldehyde\nPROCEDURE\nBe cautious at any time not to mix fluid from one well to another when pouring fluid off.\nEven a tiny bit of antibody can stain your cells.\nMake a scheme showing which antibodies in which wells. If viability test is not a part of your panel you should make a sample just with your viability marker\nAdjust your cell suspension to 1-5x 106/ml in stain buffer.\nBlocking can be performed now: Add HuIg, 100g/ml cell suspension, mix and incubate at +4 °C for at least 15 minutes.\nAdd antibodies to the wells\nTransfer 100µl cell suspension to each well and pipette up and down 5 times to mix cells and antibodies.\nIncubate in the dark 15-30 min at RT. Some cell types will require incubation 30-60 min at 4 °C\nAdd 100 µl washing buffer to each well.\nCentrifuge the plate at 350xg for 2 min at RT (or cold)\nPlace a thick paper tissue flat on the table\nPour off the supernatant in one sliding movement and press the plate briefly against the paper tissue before turning the plate bottom downwards again\nLoosen the cell pellet by a gentle knocking on the side of the plate\nAdd 200µl stain buffer to each well using a multichannel pipette, resuspend cells using the pipette.\nRepeat from 8-12 once\nThen repeat from 8-11 once (makes a total of 3 washes ending up with pellets)\nFix and resuspend the cells by adding 200µl or 250µl of fixation buffer to each well and pipette up and down 10 times\nPlace a lid on the plate and keep it cold and dark until analysis\nThe use of tandem dyes will limit the storage time to 24 hours. \u000bWe recommend analysing as quickly as possible.","cbCaiiMqosGjBTtT","https://ap.wps.com/l/cbCaiiMqosGjBTtT","docx",131055,2,"English","en",105,"# Materials\n## Stain buffer and fixation buffer\n## Blocking reagent and antibodies\n# Procedure\n## Prepare and block cells\n## Antibody incubation in the dark\n## Washing and centrifugation\n## Fixation and resuspension\n## Storage before analysis","[{\"question\":\"What is the main purpose of antibody staining in this protocol?\",\"answer\":\"To stain surface antigens on cells using a 96-well plate workflow, enabling subsequent analysis while controlling incubation, washing, and fixation steps.\"},{\"question\":\"How long should antibody incubation be performed at room temperature, and what changes if incubating on ice?\",\"answer\":\"At room temperature (RT), incubate in the dark for 15–30 minutes. If incubating on ice, expand the incubation time to 30–60 minutes and use a cold centrifuge.\"},{\"question\":\"What precautions are critical during washing to prevent incorrect results?\",\"answer\":\"Avoid mixing fluids between wells when pouring off supernatant. Use gentle handling (brief contact with tissue, gentle pellet loosen by knocking) and repeat washes as specified to obtain the correct number of wash/pellet cycles.\"}]","Antibody Staining in 96-Well Plates - 2017 | DOCX",1788166443,{"code":4,"msg":30,"data":31},"ok",{"site_id":24,"language":23,"slug":32,"title":14,"keywords":33,"description":15,"schema_data":34,"social_meta":84,"head_meta":86,"extra_data":88,"updated_unix":28},"antibody-staining-in-96-well-plates-2017","",{"@graph":35,"@context":83},[36,52,66],{"@type":37,"itemListElement":38},"BreadcrumbList",[39,43,46,49],{"item":40,"name":41,"@type":42,"position":11},"https://docshare.wps.com","Home","ListItem",{"item":44,"name":45,"@type":42,"position":21},"https://docshare.wps.com/template/","Template",{"item":47,"name":13,"@type":42,"position":48},"https://docshare.wps.com/template/forms/",3,{"item":50,"name":14,"@type":42,"position":51},"https://docshare.wps.com/template/antibody-staining-in-96-well-plates-2017/165122/",4,{"url":50,"name":14,"@type":53,"author":54,"headline":14,"publisher":56,"fileFormat":59,"inLanguage":23,"description":15,"dateModified":60,"datePublished":60,"encodingFormat":59,"isAccessibleForFree":61,"interactionStatistic":62},"DigitalDocument",{"name":9,"@type":55},"Person",{"url":40,"name":57,"@type":58},"DocShare","Organization","application/vnd.openxmlformats-officedocument.wordprocessingml.document","2026-08-31",true,{"@type":63,"interactionType":64,"userInteractionCount":4},"InteractionCounter",{"@type":65},"ViewAction",{"@type":67,"mainEntity":68},"FAQPage",[69,75,79],{"name":70,"@type":71,"acceptedAnswer":72},"What is the main purpose of antibody staining in this protocol?","Question",{"text":73,"@type":74},"To stain surface antigens on cells using a 96-well plate workflow, enabling subsequent analysis while controlling incubation, washing, and fixation steps.","Answer",{"name":76,"@type":71,"acceptedAnswer":77},"How long should antibody incubation be performed at room temperature, and what changes if incubating on ice?",{"text":78,"@type":74},"At room temperature (RT), incubate in the dark for 15–30 minutes. If incubating on ice, expand the incubation time to 30–60 minutes and use a cold centrifuge.",{"name":80,"@type":71,"acceptedAnswer":81},"What precautions are critical during washing to prevent incorrect results?",{"text":82,"@type":74},"Avoid mixing fluids between wells when pouring off supernatant. Use gentle handling (brief contact with tissue, gentle pellet loosen by knocking) and repeat washes as specified to obtain the correct number of wash/pellet cycles.","https://schema.org",{"og:url":50,"og:type":85,"og:title":14,"og:site_name":57,"og:description":15},"article",{"robots":87,"canonical":50},"index,follow",{"doc_id":7,"site_id":24},{"code":4,"msg":5,"data":90},[91,96,101,106,111,116,119,124,129],{"id":92,"doc_module":11,"doc_module_name":45,"category_name":93,"show_sort_weight":94,"slug":95},11,"Presentations",90,"presentations",{"id":97,"doc_module":11,"doc_module_name":45,"category_name":98,"show_sort_weight":99,"slug":100},12,"Resumes",80,"resumes",{"id":102,"doc_module":11,"doc_module_name":45,"category_name":103,"show_sort_weight":104,"slug":105},14,"Invoices",70,"invoices",{"id":107,"doc_module":11,"doc_module_name":45,"category_name":108,"show_sort_weight":109,"slug":110},15,"Posters",60,"posters",{"id":112,"doc_module":11,"doc_module_name":45,"category_name":113,"show_sort_weight":114,"slug":115},16,"Social Media",50,"social-media",{"id":12,"doc_module":11,"doc_module_name":45,"category_name":13,"show_sort_weight":117,"slug":118},40,"forms",{"id":120,"doc_module":11,"doc_module_name":45,"category_name":121,"show_sort_weight":122,"slug":123},18,"Letters",30,"letters",{"id":125,"doc_module":11,"doc_module_name":45,"category_name":126,"show_sort_weight":127,"slug":128},21,"Paper Templates",5,"papers-templates",{"id":130,"doc_module":11,"doc_module_name":45,"category_name":131,"show_sort_weight":4,"slug":132},158,"General","general-158"]