Rat apc conjugated anti mouse igg1

A sample of 3 × 10⁵ cells was incubated with primary antibodies in 70 μl of phosphate-buffered saline (PBS) (Sigma-Aldrich) containing 1% (v/v) bovine serum albumin (BSA) (Lorne Laboratories, Reading, Berkshire, UK) (PBSA) for 1 h with continuously mixing. The cells were washed once with PBSA and incubated with secondary antibody RPE F(ab′)₂ goat anti-mouse IgG (Dako Cytomation, Glossop, UK) in 25 μl PBAS for 30 min with continuous mixing in the dark. The cells were washed twice with PBSA and then analysed on a FC500 flow cytometer using Kaluza software (Beckman Coulter (UK) Ltd, High Wycombe, Buckinghamshire, UK). For detection of RhAG, cells stained for 30 min at 37 °C with 5 μg ml⁻¹ Hoechst 33342 (Sigma-Aldrich). Cells were incubated with extracellular antibody LA1818 against RhAG for 30 min at 4 °C, labelled with rat APC-conjugated anti-mouse IgG₁ (Biolegend, London, UK) and analysed on a MACSQuant system system (Miltenyi Biotech Ltd, Bisley, UK). ImageJ was used to quantify bands on western blot33 (link).

Cells were fixed in 1% pararformaldehyde for 15 min before permeabilisation with 0.05% Triton X-100 for 5 mins. Cells were then blocked with 4% BSA before incubation with vimentin antibody followed by rat APC-conjugated anti-mouse IgG1 (Biolegend, London, UK) and analysed on a MACSQuant system (Miltenyi Biotech Ltd., Bisley, UK).