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Centrifuge at 800 g for 30 s at room temperature Histopaque -1077 is now in the lower chamber of the tube (c) Add 10 ml RPMI-FBS solution to 5-ml bone marrow aliquots and gently mix by pipetting (d) Pipette suspension carefully to the upper chamber of the prepared Accuspin tube (e) Centrifuge at 800 g for 15 min at room temperature (f) Carefully transfer the opaque interface, which contains the mononuclear cells, into a clean Falcon tube with a pipette (g) Pellet cells by centrifugation at 300 g for 10 min and 4 C, aspirate supernate, and resuspend cells in RPMI-FBS medium (h) Count an aliquot of the cell suspension using a hemocytometer and determine viability by dye exclusion with Trypan Blue (i) Centrifuge cells at 300 g for 10 min and 4 C, aspirate supernate, and resuspend cells in expansion medium.

You might never have guessed that MythTV can also handle Webcams. In the true spirit of convergence, MythTV lets no device remain an island unto itself, and provides the glue necessary to use IP telephony and Webcam together. Plug your Webcam settings in here to further extend your MythTV experience. Webcam Device Like the capture card entry you specify for your input device, this parameter corresponds to the Webcam device as identified by Linux. Thus, if your TV capture card is already designated as /dev/video0, you should try /dev/video1 for your Webcam. Transmit Resolution Use a smaller resolution to conserve bandwidth across the network, and a higher resolution to support better-quality images at the endpoints. Transmit Frames/Second Specify the maximum number of frames that may be transmitted per second to regulate the impact streaming Webcam video imposes on your network. Valid values range from 1 30, where higher values create better-looking images at the expense of adding network overhead. Maximum Transmit Bandwidth (Kbps) Specify the maximum uplink bandwidth for your wide area network (WAN) or Internet connection. The default value here is 256 Kbps. Capture Resolution Specify the incoming resolution from the capture stream provided by the Webcam device. It is imperative that this number match your hardware specifications. You can digitally pan or zoom an image before transmission by setting a higher value here.

(j) Plate suspension at a density of 5 103 cells/cm2 in expansion medium in a cell culture ask (40 ml per ask) (k) Incubate the cells in a humidi ed 37 C, 5% CO2 incubator (l) At days 3 and 5: add 20 ml expansion medium to each ask (m) When cells reach 60% con uence: wash the cells 2 times with PBSA and add fresh expansion medium (n) Change medium twice a week until cells reach 80% con uence (days 12 22), then aspirate expansion medium and wash cells twice with PBSA at 37 C (o) Add 8 ml trypsin-EDTA to each ask and distribute the solution evenly (p) Incubate for 5 min or until cells start to detach at 37 C (q) Inactivate the trypsin by adding 8 ml control medium cooled to 4 C, mix, and transfer into a clean 50-ml Falcon tube.

I H(p1)

Use cold serum-containing medium at this step to block trypsin activity (r) Wash the ask once again by adding 8 ml control medium cooled to 4 C and combine with the mix from Step (q) in the Falcon tube (s) Centrifuge at 300 g for 10 min at 4 C (t) Aspirate the supernate and resuspend the cells in 10 ml control medium (u) Combine all cell suspensions in one Falcon tube, mix well by pipetting (v) Count an aliquot of the cell suspension using a hemocytometer and determine cell viability by dye exclusion with Trypan Blue Count unstained cells (w) Centrifuge cells at 300 g for 10 min at 4 C, aspirate the supernate, and resuspend cells at a concentration of 5 106 in FBS-DMSO solution Work rapidly as unfrozen cells should be exposed to DMSO for the shortest time possible.

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