|Species||Human (H. sapiens)|
|Tissue/Organ/Organ System||Blood Vessel|
|Applications||For Research Use Only|
|Donor Gender||Donor Info Not Disclosed|
|Isolation Method||Isolated from normal human pulmonary tissue|
|Cell Type||Primary Cells|
|Expression Profile||CD31, VE-Cadherin|
Use of PriCoatTMT25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells inECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.
The base medium for this cell line is Prigrow X series medium available at abm, Cat. No. TM104. Cells must be grown on gelatin coated plates. Gelatin Coating Solution (0.1%) is available at abm, TM063. Cells can only propagate for a maximum of 2 passages at split ratio of 1:2. Recommend to conduct media change every 3rd day to maintain optimal growth.
|Subculture Protocol||1. Remove and discard culture medium. 2. Add 2.0mL of Trypsin-EDTA(TM050) solution to flask and observe cells under an inverted microscope until cell layer is dispersed. Note: Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 3. Centrifuge cells at 1500rpm for 3 minutes to pellet. 4. Aspirate out trypsin, leaving pellet undisturbed. 5. Resuspend pellet in fresh culture medium and plate in new culture vessel. 6. Incubate cultures at 37°C.|
|Preservation Protocol||1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO.|
2. Storage Temperature: Liquid nitrogen vapour phase.
|Unit quantity||5×105 cells / 1.0 ml|
|QC||Lots are tested and are negative for HIV-1, HBV, and HCV.|
Endothelial markers are tested via immunofluorescence staining or FACS.
|Disclaimer||1. All test parameters provided in the CoA are conducted using abm‘s standardized culture system and procedures. The stated values may vary under the end-user’s culture conditions. Please verify that the product is suitable for your studies by referencing published papers. All sales are final.|
2. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a replacement is possible at a cost (plus shipping).
3. All of abm‘s cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.
4. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.
What is the supply procedure?
The frozen vial of cells will be shipped in Dry ice while T25 flasks by themselves are shipped at room temperature.
Does ''Normal" mean undiseased tissue/organ?
How often do I need to change the media?
The media should be renewed every 2-3 days
How should I store frozen vials of primary cells? Can a frozen vial be put back into liquid nitrogen after delivery?
In most cases, a vial of primary cells shipped in dry ice (-70°C) can be placed back into liquid nitrogen and recovered at a later date by rapid thawing. However it is important to note that the viability of some sensitive cell types may be reduced by the temperature shift of such treatment, making recovery more difficult. For this reason, we recommend that cells be thawed and placed into culture as soon after receipt as possible. It is best to minimize storage time at -70°C; as this is only used for shipping the cells. ABM does not warrant the viability of cells stored at -70C after shipments have been received.
How much Pen/Strep should I add to the culture media? (Only where stated in the culturing protocol provided).
We suggest 1% P/S (G255). It should not be required if your lab does not use P/S routinely however. G255 Penicillin-Streptomycin contains 10,000 units of penicillin (base), 10,000µg of streptomycin (base) per mL in WFI water. It is supplied as a 0.22 µm-filtered, 100X frozen liquid.
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at 37C, 5% CO2 and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen or dry ice; -180C
Is it possible to freeze the cells again after thawing?
Unless specified otherwise in the datasheet, primary cell lines may be re-freezed after thawing. However, primary cells are usually not good for multiple passaging and they usually last only for up to 10-12 population doublings. Our primary cells are usually provided at passage 2, therefore they can be passaged for an additional 1-2 passages. For long term use, immortalized cells are preferred.
Can I seed frozen cells directly into collagen coated 6-well plates? Or I should seed in T25 flasks (G299) first, and then transfer to 6-well plate?
If you wish to use a 6-well plate, you can seed it directly into the plate; no need to go through T25 flask then to a 6-well plate.
Why is it important to determine the optimal seeding density?
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface. Seeding density is important as many cells need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
How many cells are each vial?
The number of cells in a vial is lot-dependent. A Certificate of Analysis stating the cell quantity of the vial will be provided with your order.
Do I need to add any extra FBS other than what was provided and came with the medium kit?
No, you don’t need to add extra FBS. Simply pipette the entire content from the growth supplement container into the basal medium to make complete media. We strongly recommend you to follow the “Directions for Use” section on the datasheet for the corresponding medium kit. The same instruction can also be found on growth supplements container label.