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Featured Cells

Cell Culture Protocols

Cell Culture Protocols


General Protocol for Recovering or Freezing 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:

  • Remove and discard the cell culture media from the flask.
  • Flush the adherent layer 2 times using a 5 ml sterile pipette with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.
  • Remove and discard the wash solution from the flask.
  • Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 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 5-10 % 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. Please note that the medium should be changed every day when cells are >70% confluent to remove non-adherent cells and replenish nutrients. Pre-wash cells with 1X PBS 1-2 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.
  • 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 (Cell Biologics, Catalog No. 6914)
  • Tissue Culture Media
  • Cold Freezing Media (Cell Biologics, Catalog No. 6916).
  • Labeled Cryovials
  • Confluent cells

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  • Remove and discard the cell culture media from the flask.
  • Flush the adherent layer with a 5 ml sterile pipette 2 times with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.
  • Remove and discard the wash solution from the flask.
  • Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of 0.25% Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 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 5-10 % 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 ml freezing media per vial to be frozen.
  • Place vials 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 endothelial cells grown in a T75 flask may be frozen in 2 cryovials.
  • A confluent primary endothelial cells grown in a T25 flask may be frozen in 1 cryovial.


Flow Cytometry In Mouse Primary Endothelial Cell Analysis

General protocol for flow cytometry procedure to use an unconjugated primary antibody (2-step staining)

  • Harvest Mouse Endothelial Cells (note: you may let cells over-growing for 24-48h after cells reach confluence; it may take 3-4 days for cell to reach confluence in a T25 flask) and keep cells in the cell culture medium (M1168, freshly made Medium, 1-2 weeks) containing 1-10 ng/ml VEGF and 10% FCS for 20 min at 37°C.
  • Wash the cells 1 time with blocking buffer (1% BSA in 1X PBS with Calcium & Magnesium).
  • Adjust Mouse Endothelial Cell suspension to a concentration of at least 0.5 x 106 cells/ml in 1-2% BSA in 1X PBS with Calcium & Magnesium.
  • Keep cells in blocking buffer for 30 min at RT.
  • Add 0.1-10 μg/ml of the primary antibody (UNCONJUGATED). In general, we use 1.0 ug/ml of anti mouse CD31 antibody, Catalog No. AF3628, Anti-Mouse CD31/PECAM-1 (Polyclonal Goat IgG) from R&D SYSTEMS, INC. or CD31/PECAM-1 (Purified Rat Anti-Mouse CD31, Catalog No. 553370, BD).
  • Take 1.0 ul primary antibody from stock solution (0.2mg/ml) into 200 ul sample.
  • Incubate cells in eppendorf tubes for at least 30-45 min with gentle shaking at RT.
  • Wash the cells two times (1-1.5 ml blocking buffer) by centrifugation at 200 g for 5 minutes and resuspend them in 500 µl.
  • Add Second Antibody, Catalog No.: A-11055 (1:200 - 1:400 dilution, The Alexa Fluor® 488 Donkey Anti-Goat IgG (H+L), Invitrogen) and incubate in the dark at room temperature with gentle shaking for 30 minutes.
  • Wash the cells 2 times (1-1.5 ml blocking buffer) by centrifugation at 200 g for 5 minutes and resuspend them in 0.4 ml (5 ml Round-Bottom Tube with Cell-Strainer Cap, Catalog No. 352235 BD).
  • Keep the cells in the dark on ice and analyze the cells ASAP between 5 min - 2 hours by FACS.

CellBiologics

Primary cells differ from cell lines in many ways.  Here’s what you should know about primary cells:

  •  Primary cells have a finite life span. They are exactly like the cells that you would find in your own body, so they do die after a certain period of time in culture. The amount of time primary cells survive in culture varies according to cell type.  
  •  Primary cells can be diverse. Cancer cell lines often come from a single patient – for example, HeLa cells came from one woman, Henrietta Lacks. Primary cells, on the other hand, can come from a variety of people. Having a variety of primary cell types from many different donors is especially useful when carrying out early drug testing— it’s important to ensure that the drug is effective for everyone. Before testing the drug in human patients, researchers can use primary cells from different donors to verify that the same effects are observed. 
  • Primary cells can change in culture.  For this reason, and because primary cells do not live forever, Lifeline Cell Technology strongly suggests carrying out experiments on primary cells using earlier passages.
  • Primary cells have not been modified in any way. Except for the enzymatic and/or physical dissociation required for extracting the cells from their tissue of origin, primary cells are not altered in any way. Immortalized cell lines, on the other hand, are often transformed either with cancer genes, viruses or other inducible modifications, which could alter the outcome of experiments.
  • Primary cells are finicky. Cell lines are hardy, mostly because they have to last a long time in culture, and be capable of surviving multiple rounds of cryopreservation and thawing. Primary cells, on the other hand, tend to be more difficult to maintain in culture.

GFP Cell Preparation

·       GFP-Labeled Primary Cells employing Amaxa nucleofector transfection methods are detached from the culture flask and immediately cryo-preserved in vials. Each vial contains 0.5-1.0x106 cells per ml and is delivered frozen. GFP marked primary cells are verified by a fluorescent microscope.

·       Cells can be seeded on 30-60 mm culture dishes ready for experiments (or cells may be expanded for 1-2 passages at a split ratio of 1:2) under the cell culture conditions specified by Cell Biologics. Repeated freezing and thawing of cells is not recommended.

Protocols for GFP-labeled Primary mammalian cells   

·       Harvest the cells by trypsinization. Count an aliquot of the trypsinized cells and determine cell density.

·       Centrifuge the required number of cells (0.5-1.5 x106 cells per sample) at 220xg for 5 minutes at room temperature.

·       Resuspend the cell pellet carefully in 100 µl room temperature Nucleofector™ Solution per sample.

·       Combine 100 µl of cell suspension with 5-10 µg pmaxGFP™ Vector (recommended for initial optimization).

·       Transfer cell/DNA suspension into certified cuvette; sample must cover the bottom of the cuvette without air bubbles. Close the cuvette with the cap.

·       Select the appropriate Nucleofector™ Program.

·       Insert the cuvette with cell/DNA suspension into the Nucleofector™ Cuvette Holder and apply the selected program.

·       Take the cuvette out of the holder once the program is finished.

·       Add 500 µl of the pre-equilibrated culture media to the cuvette and gently transfer the sample immediately into the culture dish. Use the supplied pipettes and avoid repeated aspiration of the sample.

·       Incubate the cells in a humidified 37°C/5% CO2 incubator until analysis. GFP expression is often detectable after only 4 – 8 hours but ideally, cells should be left undisturbed for 24 hours. GFP expression efficiency depends on different cell types.

Transfection efficiencies: ~40-60% by using Amaxa's Nucleofector device