Rabbit anti mouse fitc

Primary antibodies used for immunoblotting were: anti-human TOP2A (mouse, MBL, 1:5000); anti-GFP (mouse, Roche, Basel, Switzerland, 1:2000); anti-HSP70 (mouse, Santa Cruz, Dallas, TA, USA, 1:4000); anti-α-tubulin (mouse, Abcam, Cambridge, UK, 1:10,000); anti-Flag M2 (mouse, Sigma, St. Louis, MO, USA, 1:4000); anti-Cyclin B1 (mouse, BD, Franklin Lakes, NJ, USA, 1:500). Secondary antibodies were: IRDye 800CW goat anti-mouse IgG (H + L) (LI-COR, 1:7000); poly-HRP goat anti-mouse (ThermoFisher Scientific, Waltham, MA, USA, 1:15,000). For indirect immunofluorescence the primary antibodies used were: anti-human topoisomerase 2α (mouse, MBL, Woburn, MA, USA, 1:500); anti-human CENP-C (rabbit, 1:1000); and anti-Flag M2 (mouse, Sigma, 1:1000). Secondary antibodies were: rabbit anti-mouse FITC (Dako, Glostrup, Denmark, 1:200); Donkey anti-mouse Alexa-Fluor 488-conjugated (ThermoFisher, 1:2000); Donkey anti-rabbit Alexa Fluor 647-conjugated (ThermoFisher, 1:2000).

Isolated CLECs and KCs were collected from passages 3 to 5. Single-cell suspensions were fixed with Fixation Reagent (Medium A; Life Technologies, Thermo Fisher Scientific, Frederick, MD, USA) for 15 min at room temperature (RT), washed with FC buffer (0.5% bovine serum albumin and 2 mM EDTA in 1× PBS), blocked with 5% goat serum in FC buffer and then immunostained with primary antibodies in Permeabilization Reagent (Medium B; Life Technologies) for 15 min at RT. Primary antibodies and their used dilution factor are shown in Supplementary Table S2. This would be followed by detection with secondary antibodies diluted with 1% goat serum in FACS buffer. Secondary antibodies used included rabbit anti-mouse FITC (1:1000, Dako, Glostrup, Denmark), Alexa Fluor 647-conjugated goat anti-rabbit (1:1000, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 647-conjugated goat anti-mouse (1:1000, Life Technologies). For fluorophore-conjugated antibodies, fixed cells were incubated with antibodies diluted in Medium B and human FcR blocking reagent (1:50, Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at RT. Stained cells were resuspended in FC buffer and subjected to analysis (MACSQuant VYB, Miltenyi Biotec) with two technical duplicates for each cell type. FC data were analyzed using MACSQuantify (Miltenyi Biotec) software based on gated live cells.

Primary antibodies used for immunoblotting were anti-human topoisomerase IIα (mbl) (1:5000), anti-human topoisomerase IIβ (BD) (1:2000), anti-GFP (Roche) (1:2000), anti-HSP70 (Santa Cruz) (1:4000), anti-myc (abcam) (1:2000) and anti-α-tubulin (abcam) (1:10 000). Secondary antibodies were IRDye 800CW goat anti-mouse IgG (H+L) (LI-COR) (1:7000) and poly-HRP goat anti-mouse (Thermo Scientific) (1:15 000). For indirect immunofluorescence, antibodies used were anti-human topoisomerase IIα (mbl) and anti-human topoisomerase IIβ (BD) (both at 1:500). Secondary antibody used was rabbit anti-mouse FITC (Dako) (1:200).