Introduction

Lactate is an intermediate product of carbohydrate metabolism. Of the two forms of Lactate, D- and L-, the L- lactate is predominant isomer found in biological systems. L-lactate is formed during the anaerobic glycolysis by conversion of pyruvate to L-lactate by lactate dehydrogenase. Lactate level is an indicator for tissue oxygen demand and utilization. Abnormally high lactate levels are associated with diseases such as diabetes and lactate acidosis. Cell Technology’s Fluoro Lactate assay is a lactate oxidase-based method for detecting L-lactate in biological samples such as serum, plasma, blood, urine, and tissue extract.

In the assay, lactate oxidase (LOX) catalyzes the oxidation of L-lactate to pyruvate, along with the concomitant reduction of hydrogen peroxide (H2O2). The detection utilizes a non-fluorescent detection reagent, which is oxidized in the presence of horse radish peroxidase (HRP) and LOX to produce its fluorescent analog.

Cell Technology’s Fluoro Lactate assay provides a reliable, sensitive fluorimetric method for the quantification of lactate in biological samples such as serum, plasma, urine, and tissue extracts.

Figure.1 Standard curve of Lactate. 50 µl serial dilutions of lactate (Starting dose 40 µM in tubes) were added to the wells of 96-well fluorescent plate. 10 µL LOX was added to each well and incubated plate at 37ºC for 10 min. 50 µL of detection reagent was added and plate was read at Ex/Em=530/590 nm.

Table 1. An example showing serum L-lactate level. 50 μl diluted serum was added to the wells of 96 –well fluorescent plate. 10 μl LOX was added to each well and incubated plate at 37ºC for 10 min. 50 μl of detection reagent was added and plate was read at Ex/Em=530/590 nm.

Table 2. Spike and recovery experiments were performed to estimate % recovery of lactate. Serum (1:100) was spiked with lactate with the concentrations mentioned in the table above. The samples were processed as described in the protocol.

Table 3. Spike and recovery experiments were performed to estimate % recovery of lactate. Serum (1:100), heat-inactivated @ 56ºC, 30 min) was spiked with lactate with the concentrations mentioned in the table above. The samples were processed as described in the protocol.

Key Benefits

• Highly effective and stable fluorescent assay for L-lactate.
• Simple and fast assay-add the reagent directly to your experimental samples. Plate can be incubated and read in 15-30 min.
• Works for serum, plasma and tissue extract.