Supernatant rinsing medium

Interleukin-8 (IL-8) levels as found in the supernatant rinsing medium (SM) in the EVEIT system of... 

VEGF levels as found in the supernatant rinsing medium (SM) of ex vivo corneas after exposure to various concentrations of SLS are shown in Fig. 5.19. The ordinate shows VEGF levels in percentage of initial values after 36 h of perfusion, i.e., immediately before the experimental exposure. The concentrations of SLS used in each set of experiments are indicated in the abscissa. Three corneas each were exposed to 1.5, 3.0, or 15.0 and 30.0% SLS (n = 3). The time scale of the analyses is explained on the right side of the diagram. 

Centrifuge these cell solutions for 5 min at 700 rpm and resuspend the pellet in culture medium for the 1st day. Seed the cells in 25-cm2 plastic flasks and incubate at 37 °C/5% C02. After 2 h of preplating (this time is required for nonmuscular cells to attach) the supernatant of this flask is filtered through a nylon mesh (pore width 100 pm) and then seeded in Petri dishes at a density of about 10,000 to 100,000 cells/cm2 in culture medium for the 1st day. After 24 h the Petri dishes are rinsed off with Dulbecco s wash solution and culture medium for the 1st... 

Monolayers of normal and FH cultured fibroblasts were incubated in the presence of [3h] - galactose in either LPDS medium or medium containing FCS. The medium was then collected and dialyzed at 4°C for 24 hrs against 4 changes of 4L water. Antibody against human serum was then added to the culture medium. Following incubation for 48 hrs at 4° the samples were centrifuged at 30,000 x g for 30 min at 4°. The supernatants were carefully withdrawn and the pellets rinsed with ice cold PBS and frozen until further use. The supernatants were freeze-dried. 

An alternative method which can also be automated by the use of the Titertek supernatant harvester (see Appendix 3) involves the measurement of radioactive chromium released into the culture medium from killed cells. The harvester consists of a set of absorbent cylinders aligned so that they may be inserted into the wells of a microtitration plate (Appendix 3). Once the supernatant in the wells has been absorbed the cylinders are transferred to counting vials and the amount of radioactive chromium released from the cell monolayer is estimated. Cells take up 51 Cr sodium chromate rapidly and the excess is readily washed away by rinsing in culture medium. 

The sample is mixed and heated to 80-90 °C on a magnetic stirrer. Then precipitation of calcium and strontium phosphates in alkaline medium (pH = 8 - 9) is performed by adding concentrated ammonium hydroxide. Precipitate is allowed to settle for at least 2 hours to one night. Then the supernatant is discarded. The precipitate is collected in a centrifuge tube and rinsed with alkalized water. Precipitate is dried in a warm atmosphere at 80°C. 

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