Figure 4

Immunofluorescence of transfected T. cruzi epimastigotes. (A) Live parasites were washed thrice with cold PBS and incubated with mAb 3F6 (anti-GP82) for 30 min on ice. Parasites were washed and incubated with 2 µg/mL Alexa Fluor 488 conjugated anti-mouse IgG antibody containing 200 nM DAPI. Samples were mounted using ProLong Gold antifade media and analysed on Olympus BX51 epifluorescence microscope. Void: epimastigotes transfected with empty pTEX plasmid; 1^st: transfected parasites carrying the pTEX-1^st construct; 2^nd: epimastigotes transfected pTEX-2^nd construct; M9/39L: transfected parasites carrying the pTEX-M9/39L construct; Control: metacyclic trypomastigotes from stationary phase cultures. (B) Fixed parasites were treated with methanol, blocked and incubated with mAb 9E10 (anti-c-myc) for 30 min on ice and processed as described above. Void: epimastigotes transfected with empty pTEX plasmid; 1^st: transfected parasites carrying the pTEX-1^st construct; 2^nd: epimastigotes transfected pTEX-2^nd construct; M9/39L: transfected parasites carrying the pTEX-M9/39L construct; Control: metacyclic trypomastigotes from stationary phase cultures. Bar: 3 μm. (C) Flow cytometry analysis of live transfected populations incubated with mAb 3F6. Live transfected epimastigotes and wild-type MT were washed and incubated with mAb 3F6 as described above. After final wash, samples were resuspended in PBS and their fluorescence levels analysed by flow cytometry on a BD Accuri C6 Flow Cytometer acquiring 10⁴ events.