Hepatocyte Counter

Counted by  _____________________________                  Hepatocyte lot no. _________________________

Date  _____________________                                              Project ID  ___________________


Trypan blue counting results

Viability calculation

Live cells            /              Total cells                     =                 Viability %
                             /                                                      =
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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


Clear cytoplasm                    
Rounded shape                  
Clear/crisp membranes             
Minimal debris                      
Swollen organelles
Membrane blebbing
Lipid droplets
Prevalent debris

Comments: _________________________________________________________________________

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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.

Overlay calculation

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 hepaticproducts@lifetech.com, 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

1. 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).


2. 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.


3. 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.


4. 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
   


5. 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.


6. 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
   


7. 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.


8. 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.