Gelatin coated dishes

Endothelial cells cultured on 35-mm-diameter gelatine-coated dish are harvested at confluence and seeded onto 60-mm-diameter gelatine-coated dishes. After 6-8 days, confluent cells are subcultured at the split ratio 1 15. Cells at the third passage can be stored in liquid nitrogen. Frozen at passage 3, brain capillary endothelial cells are re-cultured on 60-mm-diameter gelatine-coated dishes and trypsinized at confluence before seeding on filters. 

Several investigators have evaluated that fibronectin-coated substrates maintain hESC pluripotency (Table 6.1) [51-58], whereas other researchers have reported unfavorable results when culturing hESCs on fibronectin-coated substrates [31,59]. Amit et al. cultured several hESC lines (1-6,1-3, and H-9) on bovine and human fibronectin-coated dishes (5 og/cm ) in knockout DMEM (KO-DMEM) supplemented with 15% serum replacement (SR). The fibronectin-specific integrin receptor aSpi was expressed in the undifferentiated hESCs (Figure 6.7) [52]. Under these conditions, the hESCs maintained pluripotency for more than six months, whereas the hESCs cultured on gelatin-coated dishes tended to differentiate [52]. Human fibronectin was found to be relatively more favorable for maintaining the pluripotency of the hESCs compared with bovine fibronectin [52]. The applied SR contained Albumax, which is aHpid-enriched bovine serum. Therefore, this work was not performed under xeno-free culture conditions. 

For bulk differentiation of hESC/hIPSC into MESP1 /PDGFRA+ progenitor enriched cultures, we make use of an embryoid body (EB) differentiation step, which is based on a previously described protocol [14]. We pre-passage the hESC/hIPSC onto Matrigel spiked gelatin-coated dishes to facilitate colony removal for EB formation see Note 11). 

To verify that EB differentiation progressed normally from day 0 to 4 or to optimize day 1-4, a differentiation control can be used. Day 4 EB are plated on 0.1 % gelatin coated dishes in... 

Efficiency of stably integrated shRNA vectors should be confirmed in vitro. Therefore, expand two positives clones per shRNA construct on feeders, freeze aliquots, and cultivate the cells feeder-free on gelatine-coated 10 cm dishes for at least 2 days. Harvest cells and analyze the four clones for knockdown efficiency as described in steps 2 and 3 of Subheading 3.1.2. Determine the best performing clone for each siRNA target sequence. 

Remove the Gelatin solution from the newly coated dishes. 

Tumour cells are allowed to attach to gelatin-coated tissue culture dishes (see 2.4.1) when many different kinds of cells including embryonal cells grow out. These latter cells may eventually outgrow the differentiated cells and they may then be cloned (Chapter 7) (Rosenthal et al., 1970 Bernstine et al., 1973). Lines isolated in this way tend after some time to lose their ability to differentiate. 

Count the cell density and seed 20,000 cells on AlexaFluor 568 gelatin-coated MatTek dish obtained in Subheading 3.3. 

Carbodi-imide-mediated reaction with gelatin-coated tubes and petri dishes... 

Coated dishes Make fresh a 0.1% gelatin solution (Sigma) in PBS by gentle heating and shaking. Filter-sterilize while still warm. Completely cover the base of tissue-culture dishes. Leave for 2 h at room tempoature, and then aspirate off and wash IX with sterile PBS. Plate cells or cover with fiesh PBS and store at d C (maximum 2 wk). Serum-free medium 10 mL media, 20 pL N3 (serum suppl ent—see item 6). N3 serum supplement 246 pL Hank s balanced salt solution (HBSS) without calcium and magnesium, 50pL 10 mg/mL bovine serum albumin in HBSS (store at 4°C), 100 pL 100 mg/mL human transferrin in HBSS (store at -20°C), 20 pL 80 mg/mL putrescine hydrochloride in HBSS (store at -20 C), 50 pL 10 mAf sodium selenate in... 

For culturing to assay explants, cells are grown on gelatin-coated 35-mm diameter tissue-culture dishes. 

Preparation of MatTek Dish Coated with AlexaFluor 568-Labeled Gelatin... 

All the procedures of coating AlexaFluor 568 gelatin on MatTek dishes are carried out in the tissue culture hood to maintain sterility. At this incubation step, it is recommended to turn off the fan and the light to decrease uneven evaporation and photobleaching. 

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