BrdU Staining Protocol<\/p>\n
Supplies<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ethanol 100% USP (highest quality)<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 FACS Staining Buffer (1XPBS w\/ 3% calf serum, 0.05% sodium azide-filtered) \u2014Dilute staining antibodies in Buffer<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 DNase (Sigma D-5025, Bovine Pancreas)<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 RNase (Boehringer, 25 mg bovine pancreas)<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow)<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 0.15 M NaCl, 1.5 M NaCl<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 10% Paraformaldehyde (kept as stock in -80\u00b0C)<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Tween 20<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1M MgCl2<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 FACS Tubes<\/p>\n
Protocol<\/p>\n
Cells in 96-well FACS plate<\/p>\n
1.\u00a0\u00a0\u00a0 Block with 24G-2<\/p>\n
2.\u00a0\u00a0\u00a0 Surface stain cells as usual<\/p>\n
o\u00a0\u00a0 Omit\u00a0fourth channel labeled antibodies on\u00a0all\u00a0stains;\u00a0EtOH destroys APC<\/p>\n
3.\u00a0\u00a0\u00a0 Prepare tubes from which to transfer EtOH drop wise (1.2 ml EtOH on ICE)<\/p>\n
4.\u00a0\u00a0\u00a0 Resuspend cells from 96-well plate with 100 \u00b5l 0.15M NaCl (cold)<\/p>\n
5.\u00a0\u00a0\u00a0 Transfer to FACS tubes ON ICE. Add 400 \u00b5l 0.15M NaCl to each tube<\/p>\n
6.\u00a0\u00a0\u00a0 Vortex at 1\/3 speed and add EtOH with pasteur pipette at 1 drop per second.\u00a0This is a critical step…\u00a0do not add EtOH too quickly<\/p>\n
7.\u00a0\u00a0\u00a0 Incubate on ice for 30 minutes<\/p>\n
8.\u00a0\u00a0\u00a0 Spin 10 minutes @\u00a02000 RPM, 4\u00b0C<\/p>\n
9.\u00a0\u00a0\u00a0 Dump and shake liquid into waste<\/p>\n
10.Using repeat pipetter, add 1 ml FACS staining buffer into each tube<\/p>\n
11.Spin 10 minutes and dump as before (step 8)<\/p>\n
12.Add 1 ml 1% paraformaldehyde + 0.05% Tween 10<\/p>\n
For 20 ml:<\/p>\n
2.0 ml 10% paraformaldehyde 10 \u00b5l Tween 20<\/p>\n
13.Incubate at\u00a0room temperature\u00a0for 30 minutes<\/p>\n
14.Incubate\u00a0on ice\u00a0for 30 minutes<\/p>\n
15.Spin and dump as before (step 8)<\/p>\n
Add 1 ml DNase (0.15M NaCl + 4.2mM MgCl + 100 Kunitz units\/ml DNAse)<\/p>\n
For 50 ml:
\n46.5 mL dH20
\n200 ul MgCl2 (1M stock)
\n1500 uL NaCl (5M stock)
\n100 Kunitz units DNAse (volume depends on activity of batch)
\nIncubate for 30 minutes @ 25\u00b0<\/p>\n
16.Spin 10 min. and dump as before (step 8)<\/p>\n
17.Transfer cells from FACS tubes to 96-well plate. Wash once with staining medium.<\/p>\n
18.Block with 10% rat serum. Incubate 15 minute on ice. Spin and dump as before (step 8: it is critical to spin at high speed once the cells have been fixed with EtoH\/PFA since they become less dense).<\/p>\n
19.Add anti-BrdU-FITC or biotin (1:20 dilution for Phoenix flow).<\/p>\n
20.Pipette up and down to resuspend pellet. Incubate for 30 minutes on ice (or overnight at 4\u00b0C).<\/p>\n
21.Wash and dump as before. Transfer cells into FACS tubes.<\/p>\n","protected":false},"excerpt":{"rendered":"
BrdU Staining Protocol Supplies \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ethanol 100% USP (highest quality) \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 FACS Staining Buffer (1XPBS w\/ 3% calf serum, 0.05% sodium azide-filtered) \u2014Dilute staining antibodies in Buffer \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 DNase (Sigma D-5025, Bovine Pancreas) \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 RNase (Boehringer, 25 mg bovine pancreas) \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow) \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 0.15 M NaCl, 1.5 M NaCl \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 […]<\/p>\n","protected":false},"author":354,"featured_media":0,"parent":367,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-templates\/child-page.php","meta":{"footnotes":""},"class_list":["post-489","page","type-page","status-publish","hentry"],"yoast_head":"\n