Antibody Staining and Counting Fixed Dv-1 Cells That Had Been Inoculated with 2 Stocks of DiNV

The two plates were treated the same. Buffers for staining were made up here

  • Took buffers out of fridge to warm to room temp
  • Took plates out of freezer to warm to room temp
  • Dripped blocking buffer into each well of each plate that had cells until wells were half full
  • Covered plates and incubated at room temp on the bench for 30 mintues
  • Prepared the antibody soluion:
    • Diluting the antibody 1:1000
    • 25mL needed todat with 500ul per well
    • 25mL DPBS, 1% BSA, 0.1% Tween to a 50mL conical
    • 25ul primary antibody (stored in glycerol)
    • Inverted to mix
  • Dumped and tapped out plates on paper towels after 30 min incubation
  • Added 500ul antibody solution to each well of cells in each plate
  • Incubated plates in the dark in a drawer for 1 hour covered
  • After incubation dumped and tapped out plates on paper towels
  • Washed 3 times with DPBS:
    • Added DPBS to each well gently
    • Let plates sit in dark for 10 minutes
    • Dumped and tapped out plates on paper towels
  • Prepared conjugate antibody:
    • Diluting antibody 1:4000
    • 11mL needed for 200ul per well
    • Solution made in the dark and kept dark
    • 11mL DPBS
    • 2.75ul Alexa fluor 488
    • Inverted to mix
  • After 3rd wash, worked in a dark room
  • Added 200ul conjugate to each well in the plates with cells
  • Incubated plates in the drawed covered for 1 hour
  • After incubation dumped and tapped out plates on paper towels, in the dark
  • Washed 3 times with DPBS in the dark:
    • Added DPBS to each well gently
    • Let plates sit in dark for 10 minutes
    • Dumped and tapped out plates on paper towels
  • For last wash I did not dump out the DPBS
  • Wrapped plates in foil and brought upstairs to the Ackley lab to use their microscope

Counting

  • Looked at each well in the plates to find a dilution that had ~50 glowing dots to use for the FFU calculation
  • For the Passage 5 DiNV stock this was the last column, or 10^-5 dilution:
    • well 1: 32 FFU
    • well 2: 32 FFU
    • well 3: 29 FFU
    • well 4: 18 FFU
    • Average: 27.75 FFU
    • There were a lot of cells washed away in these wells but it seemed probably consistant across all the wells and plates, hard to tell. This is the issue with Dv-1 cells
  • For the Middle Band (MB) DiNV stock I used the 4th column, or 10^-3 dilution
    • well 1: 104 FFU
    • well 2: 112 FFU
    • well 3: 115 FFU
    • well 4: 91 FFU
    • Average: 105.5 FFU
  • I also took representative images of the wells I counted, the raw images in LOF format can be found here and these need to be opened with image J
  • Then I used the equation to calculate the titers of the stocks from these averages
  • Equation: (FFU average * (1000ul/ul virus added))/dilution factor of counted wells
    • note 200ul of virus solution was added to each well
  • So for Passage 5 the FFU titer came out to be: 13,875,000 FFU/mL
  • And for the Midde Band the FFU titer came out to be: 527,500 FFU/mL
  • Aliquots of these stocks are stored at -80 in my virus stock box with titers listed