Desk 2 sputter coater

HeLa cells were plated at 5×10⁵ cells per well in 6-well tissue culture plates with etched grid coverslips with imprinted numbers (BELLCO Biotechnology) 24 h prior to infection. Cells were infected with wild type Lm expressing RFP (DPL5538) at an MOI of 100 in DMEM. Bacteria were spun onto cells by centrifugation at 1500 rpm for 5 min. After 60 min of invasion at 37°C, cells were washed three times with phosphate buffered saline (PBS) with Calcium and Magnesium (Wisent #311-420-CL) followed by the addition of growth media containing 50 μg/ml Gentamicin (Wisent #400-130-IG). At 6 h post infection, cells were cooled on ice and washed twice with chilled PBS with Calcium and Magnesium. Annexin V Alexa Fluor 488 Conjugate (Invitrogen) was diluted to 1% (v/v) in chilled PBS with Calcium and Magnesium and added onto the coverslips for 10 min on ice. Cells were washed twice with chilled PBS with Calcium and Magnesium and fixed with 2.5% PFA (EM Sciences #15710) for 30 min at 37°C. Coverslips were imaged by fluorescence microscopy in PBS with Calcium and Magnesium. Subsequently, samples were fixed in 2% glutaraldehyde in cacodylate buffer, rinsed in buffer and dehydrated in a graded ethanol series. The samples were critical point dried in a Bal-tec CPD030 critical point dryer, mounted on aluminum stubs, gold coated in a Denton Desk II sputter coater and examined in an FEI XL30 SEM.