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Practice 1.
Generation of antibody-producing hybridoma cells
(The animals were immunized earlier. The number of the antigen-specific immune cells is
increased in the spleen).
1. Euthanize mouse via cervical dislocation. Remove spleen using aseptic scissors,
forceps).
2. Transfer the spleen in a 6-cm Petri dish.
- Pour 3ml medium on the organ.
- Homogenize the spleen with a spatula.
- Transfer cells to a 50ml tube, through several (~4) washes with 2-3ml medium.
Pipette the cell suspension a few times (mixing), then let sit (~4 min) until
larger tissue pieces have sedimented to the bottom of the tube.
- Collect the upper cell suspension and put in new 50 ml tube. Centrifuge at 9001000rpm for 5min.
3. Centrifuge at 300-500g (900-1000 rpm) for 10 min. Pour off the supernatant carefully.
Before resuspension, loosen pellet by finger-flicking. Resuspend cells in 10 ml
physiologic buffer solution (PBS)
4. Count the spleen cells in a Bürker-chamber (You will find the prepared, counted SP2
myeloma cells are in a separate tube).
The process of the cell counting:
- Mix 50 l spleen cell suspension + 50 l trypan blue solution in an Eppendorf
tube
- Transfer 10 l trypan blue-cell suspension to the chamber with a pipette, to fill
by capillary action. This can be done by carefully touching the edge of the
cover-slip with the pipette tip.
- Count all the living cells (living cells is not stained by the trypan blue). Count
the cells lying within a 1mm2 area, using the subdivisions (either enclosed by
three parallel lines or within 25 small rectangle). In the case of the marginal
cells, count those that lie on the top and left hand lines of each square, but not
those on the bottom or right hand lines. See Figure 1.
- Calculate the concentration applying the equation below, number of cells in
1ml
n×104×d
n = the whole number of the counted living cells in a 1mm2 area
d = the dilution (For the original cell concentration you must consider that the
volume of the cell suspension was diluted by trypan blue to twice.)
1 mm
0.2 mm
Figure 1.
1mm2 is bordered by the midst of three
parallel lines or equal to the combined area
of 25 small rectangle (16 pieces are
indicated with shading on the figure):
25×0.2mm×0.2mm = 1mm2
The distance from the cover slip: 0.1mm
The counted cells are in 0.1mm3
104 times more cells are in 1ml (1000mm3)
5. Combine 1x107 spleen cells with 2x106 myeloma cells in a 15-ml tube (ratio can
varied between 10:1 – 10:2). Fill up the tube with washing buffer. Note the counted
and combined cell numbers!!!
6. Centrifuge at 1000 rpm for 10 min.
7. Pour the supernatant. Remove liquid by an absorbent paper from the edge of the
inverted tube.
- Slowly add 1 ml PEG (37ºC) by drops to the pellet
- Loosen pellet by gentle very gentle suspending of the cells
- Slowly add 5 ml medium (37ºC) by drops and add slowly 10 ml medium.
- Incubate for 10-30 min.
8. Centrifuge at 1000 rpm for 10 min. Pour the supernatant. Add fress 10 ml medium to
the cells.
9. Chek cell fusion by microscope (Find fused cells)
10. Incubate cells in thermostat 1-4 hours (time for cariofusion in fused cells). Transfer
hybridomas at a 1-5x104 number (depending on the used myeloma cell number) into a
24 or 96-well plate in selection medium.
Cell cloning by limited dilution:
1. Count hybridoma cells.
2. Add 100 l medium to all the wells in the 96 well Add 1000-5000 cell in 100 l. Note
the transferred cell number! Then using a single channel pipette quickly transfer
100μL from the first well to well B1 and mix by gently pipetting. Avoid bubbles.
Using the same tip, repeat these 1:2 dilutions down the entire column, discarding
100μL from H1so that it ends up with the same volume as the wells above it.
3. With the 8-channel micropipette add an additional l00μL of medium to each well in
column 1 (giving a final volume of cells and medium of 200μL/well). Then using the
same pipette quickly transfer l00μL from the wells in the first column (Al through H1)
to those in the second column (A2 through H2) and mix by gently pipetting. Avoid
bubbles!
4. Using the same tips, repeat these 1:2 dilutions across the entire plate, discarding l00μL
from each of the wells in the last column (A12 through H12) so that all the wells end
up with 100μL of cell suspension.
5. Bring the final volume of all the wells to 250μL by adding 150μL medium to each
well.
6. Incubate 96-well plate at 37 ºC till colonies grow up. Single cell derived colonies are
expected at the right-bottom part of the plate.
1:2 dilutions (2)
1
A
3
4
5
6
7
8
9 10 11 12
1:2 dilutions (1)
B
C
2
D
E
F
G
H
Figure 2. 12 x 8 grid template for limiting dilution.
Bovine serum albumin-specific polyclonal antibody purification by
immunosorbent:
The goal of this part is the purification of BSA-specific antibodies from the serum of the
BSA-immunized animals. On the column, polymer (CNBr) activated Sepharose 4B) beadbounded BSA is found.
1. Drain the PBS to the top of the prepared, PBS-washed column (do not leave the
column without buffer (avoiding bubbling and deterioration of effectiveness)
- Transfer the diluted serum of the BSA-immunized animal (anti-BSA serum) to
the column.
- Allow the anti-serum get into the column. The dropping (possibly serum
containing liquid) is collected in clear tube and reintroduced to the column
again (this step can be repeated multiple times.)
- Close the column, and incubate for 30 min (antibody binding)
During the incubation time, prepare 10 tubes containing 100l 1 M pH8 TRIS
buffer and parallel 10 tubes containing 450 l Comassie Brilliant Blue (CBB)
protein stain. (Prepare an extra 5-10 tubes with CBB).
2. Wash the column with PBS (pH 7.2) (3-6 time column volume)
- Collect 1-2 drops in 450 l CBB tubes from the eluted liquid (at the bigining
and following every column volume) Samples is diluted 1:10 in the 9 time
CBB solution.
-
Wash the column till the color of the CBB solution failed to detect protein in
the eluted washing buffer. At this point no more contaminated protein is
washed from the column.
- Drain the PBS to the top of column.
3. Elute the bound specific antibodies by 4-5 ml 0.2 M glycine buffer (pH 2.3). (At this
pH, reversible conformation changes of the proteins happen and the antibodies are
released).
- Collect fractions ( 9-10 drop/fraction) in the prepared 100 l TRIS buffer
(TRIS:2-Amino-2-(hydroxymethyl)-1,3-propanediol, containing tubes pH is
neutralized in the fractions)
- Regularely collect 1 drop in a CBB tube to check protein elution. (If CBB
failed to detect protein (brown color), no more antibody can be eluted from the
column. If the color is blue (protein positive samples), continue washing with
acidic glycine buffer with some more column volume. Wash eluted clean
column with PBS, 1M NaCl and close the top of the column with 2 column
volume 1M NaCl+0.05%Na-azid buffer.
- Determinate the protein concentration of the 10 collected fractions. Add 50-50
l eluted solution into the parallel CBB tubes.
4. Combine the fractions with the highest protein content.
- Prepare a 1:2 dilution on a 96-well plate. Add 20 l PBS to all the wells of the
row A on the plate. Add 20 l from the eluted BSA-specific antibody into well
A1. Then using a single channel pipette transfer 20 μL from the first well to
well A2 and mix by gently pipetting. Avoid bubbles. Using the same tip, repeat
these 1:2 dilutions right the entire row, discarding 20 μL from A10 so that it
ends up with the same volume as the wells above it.
- Repeat the 1:2 dilution using a protein standard (highest protein content: 1
mg/ml). Do not forget to prepare a blank sample. Avoid bubbles (may cause
false positive/negative results). Eliminate bubbles with a pipette tip (or by
centrifugation).
- Add 9x volume BCC (180 l/well) to the samples. Determine the protein
content at 590 nm by photometer (Unspecific protein content can be analyzed
at 540 nm)
(Optional: dialyse antibodies overnight to remove excess salt at 4°C against 1000x
volume PBS. Use dialysis tubes. Exchange the buffer and repeat dialysis for a same
time. You can filter it through a 0.22µm filter for sterility at the end)
5. Using 1 mm graph paper, plot absorbance on the y-axis against protein concentration
on the x-axis to obtain the standard curve. Plot absorbance of the dilution of the eluted
sample and determine the protein content of the purified BSA-specific antibody
(mg/ml). You will find presentations on the department’s homepage helping in the
calculations.
Presentation:
1. Check the viability of myeloma and hybridoma cells in normal, HAT and HT medias.
2. Analysis of hybridomas in selective medium.
3. Representation of a limited dilution.
Notebook!!!!!!!!
1. Brief introduction about the selection steps of hybridoma generation. (What selection steps
are required for the generation of immortal monoclonal antigen-producing hybridoma
generation from a short lived polyclonal B cell and from a „useless” myeloma cells?
2. Antibody purification: Plotted standard curve and diluted eluted sample curve. The
calculated protein content.
3.a. Set a template using a 12x8 grid for your limited dilution. Note and calculate the cell
numbers in all the wells of your 96-well plate. (Use and calculate your noted cell number)
3.b Set a similar template as in 3.a. At this point the applied cell number in the A1 well is
100000. Calculate theoretical cell number in well H12 after the dilution steps (……..
cell/250 µl).
3.c. Consider a 96 well cell culture plate. How many single cell colonies (clones) would start
to grow, if you used the same concentration of cells in all the wells of this plate as in the
H12 before (3.b). (Suppose optimal conditions.). What volume of cell culture medium with
how many cells should be used to prepare such kind of cloning cell culture.