Kloetzel, Judith K.

Milder, Regina V.

Pinho, Rosa Teixeira de

English

Cadernos Espinosanos (E-Journal)

Rev. lnst. iVled. ìrop. São Paulo23(2) :96, março-abril, 7981MACROPHAGE.TRYPANOSOME INFEGTION. DEAD AND LIVE CELL SCORING (-)Rosa Teixeirâ, ale PINHO (1), Judith K' KLOETZEL (Z) ¿rìd Regina V' MILDER (3)During the course of our experÍments on tnernteraction between peritoneal macrophages anrlTrypanosoma cruzi in vitro, we 'weÍe interest'ed^ to observe the effect of the pârasite on thehost cell. Using the trypan blue dye exclusiontest for viability we were faced with the l'echni-cal problem of visualizing trypanosomes v/ithincells in light microscopy, without staining, theonly way to Cistinguish live from dead cells byt.his method.We here describe a modifícation of the try-pan blue dye exclusion test which overcomesthis difficulty.Nlacrclphages harvested from the perito-neum of peptone stinrulated mice were allowedto setl;le <ln coverslips, and incubated Ìor 24hours at 35'C in tissue culture medium (M199'FIow Lab.. U.K.) with 20%a letal calf serum. Try-pomastigotes of T. cruzi were obtained fromthe supernatant of LLC-MK" tissue culture in'fected cells, ancl kept rn contact with macropha-ge monolâyers for 3 hours. Monolayers werewashed and re-incubated in M 199 for 96 hours'at 35"C.Observations were carried out, washjng co-verslips in pre-lreated phosphate buffered sali'ne (PBS) and exposing them for 5 minutes toINTRODUCTIONrJ DC s93. 161 . 13a, 0.29c' trypan blue scllution in PBS' At this sta-ge Iive cells could. be distinguished from deadones, but parasites were hardl5¡ visible' Subse-quently cells were washed in PBS, fixed for 5minutes in Bouin's solution, and washed repeat-edly in 80% ethylic alcohol and further in 1000/oalcohol, xylene, and' finally mounted on slides'After processing coverslips as mentionedabove, live and dead cells could be scored, aswell as infected and non-infected cells, sinceduring fixation the blue staining of dead cellsmaintained itself while pararsites revealed theiroutline very distinctly.RESUMOfÈeconhecimenúo de células viáveis na infecçãode macrófagos Por úriPanosomasDescreve.se uma técnica, usando azul de tri-pano e posterior fixação, que permite visualiza-ção perfeita de T. arrrzi no interior de macrófa-gos e ao mesmo tempo a distinçáo entre célulashospedeiras vivas e mortas.(*) Supported by grants from Conselho Nacional d.e Desenvolvimento CientÍfico e Tecnológico - CNPq(1) Present ad.dress - Instituto Oswaldo Cruz, Rio de Janeiro, Brasil(2) Department of preventive .Medicine, Univetsity of Sáo Paulo School of Medicine, Brazil, Instituto de Medicina Tro'pical de Sáo Paulo(B) Ðepartment of pathology, University of Sáo Paulo School of Medicine, Brazil. Instituto de Medicina Tropical deSão Paulo96Recebido para publicação em 2311211980.

Macrophage-trypanosome infection. Dead and live cell scoring

https://www.revistas.usp.br/rimtsp/article/download/197045/181397

Macrophage-trypanosome infection. Dead and live cell scoring

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