Standard Lymphocyte Culture

Introduction ,[object Object],[object Object],[object Object],[object Object],------ high resolution G-banding and FISH studies ,[object Object],[object Object],[object Object],----- Under appropriate conditions, active division of lymphocyte can be induced in vitro

Stimulation of lymphocytes and the use of mitogens ,[object Object],[object Object],[object Object],[object Object],[object Object],In vivo: Antigens ---> small to enlarge ---> non-dividing to proliferation and division In vitro: Mitogens --->PHA ( a mucoprotein) or PWM transforming large populations of lymphocytes into mitotically active cells.

Standard lymphocyte culture PHA exposure ------> morphological transformation ------> RNA synthesis, nuclei enlarge and DNA synthesis commences ------> wave of mitotic activity 48, 72 and 96 hr 1. Collection, transport and storage of samples Adult or older children: 5-10 ml with heparin Fetal and neonatal samples: 1-2 ml with heparin Samples are stable for several days

Culture medium: constituents and storage 2. Culture medium: constituents and storage ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Initiation of a routine 72-h culture ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Harvesting lymphocyte culture The point of harvest is engineered so that a maximum number of cells are in metaphase. ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Protocol for harvesting lymphocyte cultures ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Thymidine synchronization of cell division Since all cell do not react to PHA at the same time, this synchrony of cells division is fairly inexact > 1 mM The effect of excess thymidine is to greatly increase the proportion of cells in S-phase

Thymidine synchronized lymphocyte culture ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

FDU ( f luoro d eoxy u ridylate) and MTX ( m etho t re x ate) Foloic acid cycle MTX FDU

Slide making ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Factors affecting slide making ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Standard Lymphocyte Culture

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