Data shown are mean values??1 s.d. IDO-IN-3 [and [ for 6,000 and 10,000?rpm (and and is given by: is the permeate volume over interval (equal to is the volume of the retentate chamber. Hence:
(10) This parameter is calculated at 5\min intervals using the LDH readings from the permeate. Therefore, to simplify equation (10), the proportion of intracellular LDH (that of intact cells) remaining in the USD membrane separation device, , versus time can be monitored and is given by:
(11) Figure ?Figure55 is a stacked bar chart which shows the measured amount of both total and extracellular LDH, as well as the calculated intracellular LDH for each of (a) the feed, F; (b) control, C; and (c) retentate post\processing, PP, at 6,000 and 10,000?rpm. The cumulative amount of soluble LDH in the permeate stream, PLDH, is also IDO-IN-3 shown on the post\processing samples. Important information may be acquired from the interpretation of this figure such as: (i) there is no significant difference in the total LDH present in the feed and the non\sheared control held for 60?min; (ii) there is good agreement in the amount of total LDH in the feed and that after processing. The first observation is of relevance to show that LDH was stable during the period of time measured and that the release of LDH is due to the effect of processing conditions and IDO-IN-3 not an artifact of experimental procedure. These observations are in agreement with previous studies carried out by Berger and Tietz (1976) and Goldblum et al. (1990) and confirm that there is no loss of LDH activity by merely holding the sample without processing. Goldblum et al. (1990) measured LDH activity in insect cells every 30?min for 3?h showing no significant changes during this period of time. Moreover, Berger and Tietz (1976) reported LDH in serum to be stable for at least 3 days at room temp. Open in a separate window Number Rabbit Polyclonal to RAB3IP 5 Amount of LDH measured and expected for the feed (F), control (C), and post\processing (P) samples at 6,000 and 10,000?rpm disc speeds (? maximum??1.9 and 13.5?W?mL?1, respectively). The bars represent the cumulative LDH measured in the permeate stream (), the measured soluble or extracellular LDH (?) and the expected internal LDH (). The individual points (? 6,000 rpm and ? 10,000 rpm) represent the total LDH (sum of permeate, extracellular and internal). All experiments were carried out at a concentration of 2??106 total cells mL?1. The control is definitely a non\sheared sample held in a centrifuge tube concurrently, 21??1oC, for the duration of the experiment. Large disc rate resulted in an increased amount of LDH measured in the permeate compared to low rate and, therefore, a decreased amount of expected internal LDH. Data demonstrated are mean ideals??1 s.e. (6,000?rpm j?=?4 and n?=?4; 10,000?rpm j?=?5 and n?=?4). Overall, from your LDH data in Number ?Figure55 it is evident that processing at high disc speed for 60?min results in an increased amount of LDH measured in the permeate compared to low disc rate. The next section addresses the results by analysis of cell damage with time of operation for each individual run as well as the average of the five repeats at low and high disc speeds. IDO-IN-3 It will also include an analysis within the styles observed with the trypan blue exclusion data. The Effect of Disc Rate (Maximum Energy Dissipation Rate) on Loss of Intact HCA2 Cells Studies to IDO-IN-3 evaluate the effect of disc rate on loss of intact cells were carried out for low (6,000?rpm) and large (10,000?rpm) disc speeds. These two speeds are equivalent to 1.9 and 13.5?W?mL?1maximum energy dissipation rates, respectively. The major increase with higher rate is due to an enhanced axial flow blood circulation effects. The circulation characteristics in the USD device will range from undeveloped laminar to turbulent circulation while at full.