Counted by _____________________________ Hepatocyte lot no. _________________________
Date _____________________ Project ID ___________________
Trypan blue counting results
|Quadrant||Live cells||+||Dead cells||=||Total cells|
Live cells / Total cells = Viability %
Total yield calculation
Total cells / Quadrants counted x Dilution factor x 10,000 x Current volume (mL) = Total cell yield
/ x 2 x 10,000 x = x 106 cells
Cell morphology notes
Seeding density calculation
Total cell yield / Desired density* = Total volume needed
x 106 cells / x x 106 cells/mL = mL
Total volume needed -- Current total volume = Volume to add to cell stock
mL -- mL= mL
* Refer to supplier recommendations. Desired density for human plated hepatocytes is typically 0.75 x 106 cells/mL, 0.8 x 106 cells/mL, or 0.9 x 106 cells/mL for a 24-well plate. Densities for suspension use or other species will vary.
Total volume of 4°C medium needed* x Final gel concentration / initial protein concentration† = Volume of gel stock to add to 4°C medium
mL x 0.35 mg/mL / mL = mL
* Medium needs to be kept at 4°C on ice. We recommend Williams Medium E (Life Technologies Cat. No. CM6000) combined with a serum-free Maintenance Supplement Pack (Life Technologies Cat. No. CM4000).
† Refer to supplier’s specification sheet. We recommend Geltrex™ Reduced Growth Factor Basement Membrane Matrix (Invitrogen Cat. No. 12760-021) at a concentration of 0.35 mg/mL.
Need assistance? For technical support on hepatocyte products, visit us at www.lifetechnologies.com/hepatocytes,
email us at firstname.lastname@example.org, or contact us by phone at: +1 866 952 3559 (US toll-free); +1 919 237 4500 (toll); +44 (0)141 814 5900 (Europe).
Advance preparation for cryopreserved hepatocytes
Note: Use universal safety precautions and the appropriate biosafety cabinet when handling primary hepatocytes.
- Thaw cryopreserved hepatocytes quickly (<2 min) in a 37°C water bath and dilute cells immediately in 37°C thawing medium. Rinse the cryovial with thawing medium to remove all cells, then centrifuge at low speed to remove cryoprotectant (refer to vendor instructions). For example, thawed human hepatocytes should be quickly transferred to 40 mL of 37°C CHRM® Medium (Life Technologies Cat. No. CM7000) and centrifuged at 100 x g for 10 min.
- Gently resuspend hepatocytes to a concentration of approximately 106 cells/mL using serum-containing medium that has been prewarmed to 37°C. For hepatocytes that will be plated onto collagen-coated plates, we recommend Williams Medium E (Life Technologies Cat. No. CM6000) supplemented with serum-containing Plating Supplement Pack (Life Technologies Cat. No. CM3000).
Preparation of the cell counting vial
- Mix 200 μL buffer (PBS or similar) or medium with 50 μL 0.4% trypan blue stain (Cat. No. T10282) in a 2.0 mL round-bottom microcentrifuge tube to make a stock trypan blue solution. Transfer 50 μL of this diluted trypan blue solution into another 2.0 mL vial, which will be used for counting the cells (this is the cell counting vial).
- Before adding hepatocyte suspension to the counting vial, gently invert the tube of hepatocytes several times to ensure a homogeneous suspension. This step is crucial to obtaining an accurate cell count.
- Using a wide-bore tip, pipet 50 μL of the hepatocyte suspension into the cell counting vial by drawing the suspension into the pipette tip once and dispensing the contents into the cell counting vial. Do not rinse out the tip. This results in a 2-fold dilution of the original cell suspension.
- Gently flick/tap the counting vial to ensure a homogeneous mixture. Do not shake or agitate vigorously, as this will cause cell death and result in an inaccurate viability determination.
Preparation of the hemocytometer
- Wait one minute, gently flick/tap the counting vial again to mix, and then slowly pipet 10 μL of the mixture into the V-shaped groove on one side of the hemocytometer. Capillary action will pull the cell suspension into the hemocytometer.
- Before counting, quickly scan the quadrants under the microscope to ensure cells are evenly distributed. If the hemocytometer is loaded unevenly or contains a bubble, reload.
Counting the hepatocytes
- Immediately count the viable hepatocytes (clear or yellow color) and dead hepatocytes (blue color) in the four outer quadrants of the hemocytometer, including any cells on two of the outer quadrant lines (Figure 1). Do not delay in counting, as prolonged exposure to trypan blue can result in cell death.
- Record the results on the reverse side of this page, and perform the indicated calculations to determine the total number of viable hepatocytes. Use additional supplemented plating medium or equivalent to dilute the hepatocyte mixture to the desired final cell concentration.
Figure 1. Counting cells on a hemacytometer.
Count all four quadrants after loading the cell-trypan blue mixture onto one side of the hemacytometer (circled in B). When counting cells on the lines of the quadrants, two edges should be included in the cell count, and two edges should be excluded. In drawing C, for example, count the cells touching the lines on the top and left sides (green circles), but do not count the cells on the lines on the bottom and right sides (red circles).