Retinoic acid incubating with cells

We recommend that the incubation of cells with radiolabeled retinoic acid is performed in the absence of serum Retinoic acid binds with low affinity to serum albumin (11) and this may reduce the concentration of free retinoic acid available for entry into the cells. For studies with [ H] ll-trans retinoic acid (15-50 Ci/ mmol), we have obtained satisfactory results using a final concentration of 20 nM. In the presence of 10% (v/v) fetal calf serum (FCS), we find that it is necessary to increase the retinoic-acid concentration to 0 2 pM to obtain a similar level of binding of retinoic acid to nuclear receptors... 

FIGURE 3.2 Effect of retinoic acid (RA) and various lipids on cell differentiation of HL-60 cells (Tochisawa et al., 1997). HL-60 cells (5xl04) were incubated with the individual lipids for 24h, then RA was added followed by incubation for additional 72h. Nitroblue tetrazolium (NBT) reducing activity in HL-60 cells was assayed. Data shown as mean value SD. p<0.01 versus control. p<0.01 versus RA. 

Incubate 5 x 10 -1 x 10 cells/mL HL60 cells for 3-6 days with 1 pM all-fran5-retinoic acid (7) or 1.25% DMSO (8). After 6 days of exposure, approximately half of the cells will appear granulocytic and show myeloperoxidase staining see Note 7). 

The alkylation-induced changes had demonstrated that important acceptors like histone HI may become nearly exclusively mono(ADP-ribosyl)ated. Mono(ADP-ribosyl)ation of proteins also appears to be of importance in certain diffemtiation processes. When F9 teratocarcinoma cells were incubated with low concentrations of retinoic acid, they differentiated into endoderm cells [22]. Under these conditions, mono(ADPR) conjugates increased considerably. The increase was time dependent, and it was mainly due to a fraction that was neither carboxyl- nor arginine-linked, nor did it correspond to nonenzymic conjugates. However, further experiments are required to determine the nature of these conjugates. 

To isolate RARs, we routinely incubate cultured cells with radiolabeled retinoic-acid isomers, prepare nuclei, extract the ligand-bound receptors from the purified nuclei and fractionate them using either sucrose density-gradient centrifugation or high-performance size-exclusion chromatography (HPSEC). It is also possible to prepare nuclear extracts from untreated cells and incubate this material with radiolabeled retinoic acid prior to fractionation (3). How-... 

Incubation of cells with radiolabeled retinoic acid is best carried out in serum-free medium (see Note 2). 

Incubate the cells with the radiolabeled retinoic acid for 2-4 h at 37°C It is important that the incubation should not be prolonged, because retinoic acid may be metabolized quite quickly (see Note 3)... 

The specificity of binding of radiolabeled retinoic acid to nuclear proteins can be tested by incubating the cells with the radiolabeled retinoic acid in the presence of 100-fold excess of unlabeled retinoic acid. 

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