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人皮肤成纤维细胞系HFF-1
HFF-1Cat# NTCC925274Product categoryHuman cellsOrganismHomo sapiens, humanCell typefibroblastMorphologyfibroblastTissueSkin; ForeskinDiseaseNormalApplicationsStem cell researchGeneralSpecific applicationsCan be used to produce feeder cellsCharacteristicsGrowth propertiesAdherentDerivationThe cell line was established in 2003 from normal human foreskin pooled from two individuals.AgeneonateGenderMaleCommentsThe growth of these cells should be arrested before being used as a feeder layer. ATCC has successfully irradiated (ATCC SCRC-1041.1) and treated the cells with Mitomycin C (NTCC SCRC-1041.2) for use as a feeder layer. If the HFFs are being used as a feeder layer for ES cells, it is not recommended to use them past PDL 45. It is recommended that the feeder cells be plated 24 hours before use at 5 x 104 cells/cm2 in order to obtain a supportive monolayer for stem cell growth.Handling informationUnpacking and storage instructionsCheck all containers for leakage or breakage.Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below _x001f_-130°C, preferably in liquid nitrogen vapor, until ready for use.Complete mediumThe base medium for this cell line is NTCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium:fetal bovine serum to a final concentration of 15%This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended).Temperature37°CAtmosphere95% Air, 5% CO2Handling procedureIt is not necessary to coat flasks with gelatin prior to plating cells, if tissue culture quality flasks are used. To insure the highest level of viability, be sure to warm media to 37°C before using it on the cells. Cells should be plated at a minimum cell density of 0.8X104 cells/cm2.Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.Transfer the vial’s contents plus 5 mL of complete growth medium to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete growth media to bring the total volume to 10 mL.Gently mix and pellet the cells by centrifugation @ 270 xg for 5 minutes.Discard the supernatant and resuspend the cells with 10 mL fresh growth medium (warm) and plate the cells at seed density of 0.8 X104 cells/cm2.Add more fresh growth medium (warm) to obtain the total volume recommended for the flask.Incubate 37°C in a 5%CO2 in air atmosphere.Fluid change twice a week or when pH decreases. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).Subculturing procedureProcedure:To insure the highest level of viability, be sure to warm media and Trypsin/ EDTA to 37ºC before using it on the cells.Cells should be split when they reach confluency. A split ratio of 1:5 to 1:7 is recommended. Volumes used in this protocol are for 225 cm2 (T225); proportionally reduce or increase amount of dissociation medium for culture flasks of other sizes.Remove and discard culture medium.Briefly rinse the cell layer with 1X PBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.Add 5 mL of Trypsin-EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to flask and incubate for 1 minute, gently tapping the flask observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).Add 6.0 to 8.0 mL of complete growth medium and rinse surface of the flask to detach all cells. Gently pipetting up and down will break cell clumps.Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 xg for 5 minutes.Remove and discard the supernatantAdd 10 mL complete growth medium to cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).Add more complete growth medium to cell suspension as needed to plate cells at approximately 5x106/T225 flask.Place flasks in incubator @ 37°C with a 5% CO2 in air atmosphere.Flask/Plate Growth Area (cm2) 1xPBS (mL) Trypsin/EDTA (mL) Equal vol. Complete Growth Medium (mL) Growth Medium (mL) T225 225 10 ± 0.2 6 ± 0.2 6 ± 0.2 30 75 75 5 ± 0.1 3 ± 0.1 3 ± 0. 1 12 T25 25 3 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 6 6 well 9.5 1 ± 0.1 1 ± 0.1 1 ± 0.1 3 Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 5th edition, published by Alan R. Liss, N.Y., 1994. Subcultivation Ratio: A subcultivation ratio of 1:5 to 1:7 is recommendedMedium Renewal: Twice a week or as pH decreasesReagents for cryopreservationDulbecco’s Modified Eagle’s Medium 30-2002, 7% FBS, 10% (v/v) DMSO. Lots produced prior to May 2019 may have used a different cryopreservation medium (complete growth medium supplemented with an additional 40% FBS and 10% (v/v) DMSO), contact Technical Support for further details.
Supplier来源:BioVector NTCC Inc.
TEL电话:400-800-2947
Website网址: http://www.biovector.net
HFF-1Cat# NTCC925274Product categoryHuman cellsOrganismHomo sapiens, humanCell typefibroblastMorphologyfibroblastTissueSkin; ForeskinDiseaseNormalApplicationsStem cell researchGeneralSpecific applicationsCan be used to produce feeder cellsCharacteristicsGrowth propertiesAdherentDerivationThe cell line was established in 2003 from normal human foreskin pooled from two individuals.AgeneonateGenderMaleCommentsThe growth of these cells should be arrested before being used as a feeder layer. ATCC has successfully irradiated (ATCC SCRC-1041.1) and treated the cells with Mitomycin C (NTCC SCRC-1041.2) for use as a feeder layer. If the HFFs are being used as a feeder layer for ES cells, it is not recommended to use them past PDL 45. It is recommended that the feeder cells be plated 24 hours before use at 5 x 104 cells/cm2 in order to obtain a supportive monolayer for stem cell growth.Handling informationUnpacking and storage instructionsCheck all containers for leakage or breakage.Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below _x001f_-130°C, preferably in liquid nitrogen vapor, until ready for use.Complete mediumThe base medium for this cell line is NTCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium:fetal bovine serum to a final concentration of 15%This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended).Temperature37°CAtmosphere95% Air, 5% CO2Handling procedureIt is not necessary to coat flasks with gelatin prior to plating cells, if tissue culture quality flasks are used. To insure the highest level of viability, be sure to warm media to 37°C before using it on the cells. Cells should be plated at a minimum cell density of 0.8X104 cells/cm2.Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.Transfer the vial’s contents plus 5 mL of complete growth medium to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete growth media to bring the total volume to 10 mL.Gently mix and pellet the cells by centrifugation @ 270 xg for 5 minutes.Discard the supernatant and resuspend the cells with 10 mL fresh growth medium (warm) and plate the cells at seed density of 0.8 X104 cells/cm2.Add more fresh growth medium (warm) to obtain the total volume recommended for the flask.Incubate 37°C in a 5%CO2 in air atmosphere.Fluid change twice a week or when pH decreases. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).Subculturing procedureProcedure:To insure the highest level of viability, be sure to warm media and Trypsin/ EDTA to 37ºC before using it on the cells.Cells should be split when they reach confluency. A split ratio of 1:5 to 1:7 is recommended. Volumes used in this protocol are for 225 cm2 (T225); proportionally reduce or increase amount of dissociation medium for culture flasks of other sizes.Remove and discard culture medium.Briefly rinse the cell layer with 1X PBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.Add 5 mL of Trypsin-EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to flask and incubate for 1 minute, gently tapping the flask observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).Add 6.0 to 8.0 mL of complete growth medium and rinse surface of the flask to detach all cells. Gently pipetting up and down will break cell clumps.Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 xg for 5 minutes.Remove and discard the supernatantAdd 10 mL complete growth medium to cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).Add more complete growth medium to cell suspension as needed to plate cells at approximately 5x106/T225 flask.Place flasks in incubator @ 37°C with a 5% CO2 in air atmosphere.Flask/Plate Growth Area (cm2) 1xPBS (mL) Trypsin/EDTA (mL) Equal vol. Complete Growth Medium (mL) Growth Medium (mL) T225 225 10 ± 0.2 6 ± 0.2 6 ± 0.2 30 75 75 5 ± 0.1 3 ± 0.1 3 ± 0. 1 12 T25 25 3 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 6 6 well 9.5 1 ± 0.1 1 ± 0.1 1 ± 0.1 3 Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 5th edition, published by Alan R. Liss, N.Y., 1994. Subcultivation Ratio: A subcultivation ratio of 1:5 to 1:7 is recommendedMedium Renewal: Twice a week or as pH decreasesReagents for cryopreservationDulbecco’s Modified Eagle’s Medium 30-2002, 7% FBS, 10% (v/v) DMSO. Lots produced prior to May 2019 may have used a different cryopreservation medium (complete growth medium supplemented with an additional 40% FBS and 10% (v/v) DMSO), contact Technical Support for further details.
Supplier来源:BioVector NTCC Inc.
TEL电话:400-800-2947
Website网址: http://www.biovector.net
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