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NEWS


You can now order Cell Biologics' products through Fisher Scientific. Search by Cell Biologics' product catalog number or key words...

In July 2017

In June

  • Lymphatic Forum, June 8-10, 2017, Chicago, IL
  • Biogen Idec | Novartis | MIT | Whitehead Institute, June 21, 2017, Boston, MA
  • Mayo Clinic, June 28, 2017, Rochester, MN 

in May

Phone: 312-226-8198

Fax:      312-226-8958

E-mail: info@cellbiologics.com

PROMOTIONS


  • Get Free Gelatin-Based Coating Solution per primary cell order,  which can be used for most primary cell culture from CellBiologics through 2017.

NEW PRODUCTS


Culture Primary Cells

Protocol - Culture Primary Cells 

All cell culture procedures must be conducted in a bio-safety cabinet.

Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.

Use aseptic technique to prevent microbial contamination.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival.

Medium:

Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements).  Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells and when changing media or splitting cells.

Coating of flasks or dishes:

Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950) for 10-30 min and then aspirate the excess solution before seeding cells.

Cell recovery from cryovial:

  • Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
  • Promptly remove the vial and wipe it down with 70% ethanol.
  • Transfer cells from the vial to a sterile centrifuge tube. Add 8-10 ml of pre-warmed Cell Biologics Cell Culture Medium. Flush the vial with an additional 0.5-1 ml of medium to ensure complete transfer of cells to the centrifuge tube.
  • Centrifuge cells at 200 g for 5 minutes.
  • Aspirate the supernatant and resuspend the cell pellet in 6 ml of Cell Biologics’ Cell Culture Growth Medium.
  • Add resuspended cells into a T25 flask pre-coated with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950).
  • Place the T25 flask in a humidified, 5%-CO2 incubator at 37°C.
  • Change culture media the following day to remove non-adherent cells and replenish nutrients.
  • Change cell culture medium everyday when cells are >70% confluent.
  • Cells should be checked daily under a microscope to verify appropriate cell morphology.

Expansion of cultured primary cells:

  • Flush the adherent layer with a 5 ml sterile pipette 3 times to dislodge loosely attached cells.
  • Remove and discard the cell culture media from the flask.
  • Wash adherent cells 2 times with 10 ml of sterile PBS (1X) without calcium and magnesium to remove nonadherent cells or fraction.
  • Remove and discard the wash solution from the flask.
  • Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA (1X) solution (Invitrogen catalog number 25300) for 1 minute. Use 2.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from T75 flasks, and 1.0 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 8-10 ml of Cell Biologics Cell Culture Medium supplemented with 5-15 % FBS to a T25 or T75 flask (the FBS will neutralize the trypsin).
  • Plate cells in fresh flasks or plates precoated with Gelatin-Based Coating Solution in a humidified, 5%-CO2 incubator at 37°C.
  • Change culture media the following day to remove non-adherent cells and replenish nutrients.
  • Cells should be checked daily under a microscopy to verify appropriate cell morphology.
  • Change culture medium every 24-48 hours. The medium should be changed every day when cells are >70% confluent to remove non-adherent cells and replenish nutrients.
  • Pre-wash cells with1X PBS 2 times whenever replacing the medium.

We recommend splitting primary cells at the follow ratio:

  • The recommended split ratio for primary murine cells is 1:2 (Smooth Muscle Cells, Fibroblasts, Endothelial Cells ) or 1:3 (Epithelial Cells, Endothelial Cells, Fibroblasts).
  • A confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Procedure for Freezing Cells

Materials:

  • 1X Phosphate Buffered Saline (PBS-1X)
  • 0.05% or 0.25% Trypsin-EDTA (1X) solution (Invitrogen catalog number 25300)
  • Tissue Culture Media
  • Cold Freezing Media (10% DMSO, 20% FBS and 70% culture medium)
  • Labeled Cryovials
  • 90-100% Confluent cells

          ----------------------------------

  • Flush the adherent cell layer with a 5 ml sterile pipette 3-5 times to dislodge loosely attached cells.
  • Remove and discard the cell culture media from the flask.
  • Wash adherent cells 2-3 times with 10 ml of sterile PBS (1X) without calcium and magnesium to remove nonadherent cells or fraction.
  • Remove and discard the wash solution from the flask.
  • Incubate cells with warm (37°C) Trypsin-EDTA (1X) solution for 1 minute. Use 2.0-3.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from T75 flasks, and 1.0-1.5 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 10 ml of Cell Culture Medium supplemented with 5-15 % FBS to the flask (the FBS will neutralize the trypsin).
  • Centrifuge the cell suspension at 200 g for 5 minutes.
  • Remove supernatant with sterile Pasteur pipette.
  • Quickly re-suspend pellet by adding 1.0 ml freezing media per vial to be frozen.
  • Place Cryovials in Nalgene "Mr. Frosty" freezing container containing100% isopropyl alcohol at -70-80 °C for 24 h.
  • Transfer vials to liquid N2 tank for indefinite storage.

We recommend freezing primary cells at the follow ratio:

  • A confluent primary cells grown in a T75 flask may be frozen in 2 or 3 cryovials.
  • A confluent primary cells grown in a T25 flask may be frozen in 1 or 2 cryovial.

Cell Biologics