Protocols

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For Human and Animal Blood and Bone Marrow Derived Cells

1.       Remove the vial of cells from dry ice (upon receipt of shipment), from the -152°C freezer or the gaseous phase of liquid nitrogen and place vial in a 37°C water bath for 1 minute or until the cells begin to thaw. Ensure that the vial is not completely submerged and water does not enter the vial.

2.       Remove vial from the water bath and wipe the outside with 70% isopropanol or ethanol to sterilize.

3.       In a Class II Biosafety Cabinet, using a 2 mL pipette, transfer cells into a 15 mL tube containing 10 mL of IMDM + 10% FBS or appropriate culture medium.[1] (all work inside the hood should be performed with sterile instruments and reagents)

4.       Use 1 mL of IMDM + 10% FBS or appropriate culture medium to wash the vial. This ensures that any remaining cells in the vial are recovered. Add rinse volume to the 15 mL tube.

5.       Centrifuge the 15 mL tube containing cells at 400 x g for 8 minutes.

6.       After centrifugation, decant and discard supernatant. If the cells will not be usedimmediately after resuspension, add DNase at 1μg/mL final to prevent cells from clumping.

7.       Resuspend the cell pellet in a small volume of IMDM + 10% FBS or appropriate culture medium (~1 mL) and measure the total volume of cells (total volume is required for cell recovery and viability calculations).

8.       Perform a viable cell count using glacial acetic acid and trypan blue.[2]

9.       Suspend cells at the appropriate concentration for your specific assay.

[1] When thawing >10 million cells, transfer cells into a 50 mL tube containing 40 mL of IMDM + 10% FBS or appropriate culture medium.

[1] Manual Cell Count is recommended for accuracy for viability determination, please see “Instructions: Manual Enumeration of Cells” included in your shipment. Propidium iodide staining by FACS is not as accurate and is not recommended.

ReachBio Research Labs does not guarantee cell viability results using alternate thawing procedures.

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For Counting Human and Animal Blood and Bone Marrow Derived Cells

  1. Prepare a 1:10, 1:20 or 1:200 dilution of cells in 3% [0.5 M] acetic acid in a tube or a well of the 96 well plate (for a 1:10 dilution dispense 10 µL of the sample into 90 µL of acetic acid).
  2. Prepare the same dilution as step one in trypan blue in a tube or a well of the 96 well plate.
  3. Clean the hemocytometer and place the cover slip over the gridded areas.
  4. Dispense approximately 5-7 µL of the acetic acid cell suspension onto one side of the hemacytometer. On the other side of the hemocytometer place 5-7 µL of the trypan blue cell suspension and let stand for approximately 20 seconds.
  5. Place the hemocytometer on the light microscope.
  6. Starting with the acetic acid cell suspension, enumerate the cells within a certain number of squares until a minimum of 100 cells are counted.[1]
  7. Enumerate the cells in the trypan blue cell suspension in the same number of squares as in step 6. In addition, determine the number of white cells and blue cells in the trypan blue cell suspension.
  8. Compare the total cell counts between the two squares – this will determine if accurate pipetting was performed in the generation of the cell suspensions.  If the total cell numbers are within 15% of each other, proceed with the cell count determination.  If the variability in cell number is greater than 15%, start the procedure again.
  9. To determine the cell count, the following equation can be used:[2]

cells/ml=(average cells per square)(dilution factor, i.e.10, if 1:10 was used)(104)

10. To determine the percent of viable cells in the sample, divide the white cell number from the trypan blue count by

the total cell number (blue and white) and multiply by 100%.

 

 


[1] If more than 100 cells are counted in one large square, the cell suspension is too concentrated and will need to be diluted to get an accurate cell count.  If less than 50 cells are enumerated in the total of 9 large squares, the cell suspension is not concentrated enough to get an accurate cell count and a different dilution needs to be prepared).

[2] Each of the 9 squares of the hemacytometer with cover slip in place represents 0.1 mm3 (or 10-4 cm3) which is equivalent to 10-4 mL.

 

ReachBio Research Labs recommends the use of manual enumeration when using single cell or purified populations.