User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/12

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Counting cells Main project page
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Summary

To ensure proper comparison of results is possible it is advisable to work with comparable amount of cells in different samples. For this it is required to count cells and calculate how many should be added to new wells.
For 24-wells (500 μL) 30.000 cells per well should be used
For Ø 10 cm petridishes (10 mL) 1×106 cells per plate
For 6-wells (2 mL) 200.000 cells

Materials & Methods

Materials

  • DMEM S+
  • 24-wells plate
  • Petri dishes

Method

  • Perform as in splitting cells
  • In stead of dividing the 40 mL over 4 plates count cells using Bürker-Türk counting chamber (See image 120220101)
    • Put ~10 μL in each chamber and count the cells present in 4 large squares, selected randomly from both chambers.

http://upload.wikimedia.org/wikipedia/commons/f/f2/Neubauer_improved_schema.gif

Image 120220101: Bürker-Türk counting chamber; Each chamber consists of 4 big blocks, like the square shown here in centre. Each big block consists of 16 small blocks (0.04 m2. The 4 big blocks are separated from the rest by 3 lines. Each row of squares is separated from the other by 2 lines (picture shows 3). Cells are counting within the 16 smaller squares and the spaces in between the rows. However only 2 of the 4 boundary spaces represented by the 3 lines are counted.

  • The average of cells in the 4 blocks has to be multiplied by 104 (amount of cells per mL) and then by the volume in which the cells are resuspended (40 mL).
  • Clean the counting chamber with 70% EtOH
  • Correct the amount of cells by diluting with DMEM S+
    • Amount of cells per 40 mL required is 2.4×106 cells
  • Divide cells over 24-wells plate
Caption
1 2 3 4 5 6
A
B
C
D
  • Prepare 2 Petri dishes as a new passage to keep the line going.

Results

  • Cell count
    • 32 cells in block 1
    • 54 cells in block 2
    • 30 cells in block 3
    • 20 cells in block 4
  • time 104 and dived by 4 gives 33.25×104 cells per mL
  • For 2.4×106 cells
    • 7.2 mL cell suspension
    • 32.8 mL DMEM S+
  • For plates
  • 14 mL DMEM S+
  • 6 mL cell suspension

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