Protocols and Lab Notebook
1. Culture of Bladder Cancer cell line
Cell culture Protocol
Bladder Cancer cells were purchased from Tedor bilharziasis institute, Cairo, Egypt, cells were cultured using DMEM (Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone), 10 ug/ml of insulin (Gibco), and 1% penicillin-streptomycin (Gibco). All of the other chemicals and reagents were from Gibco- Germany; Invitrogen.
Plate cells (cells density 1.2 – 1.8 × 10,000 cells/well) in a volume of 100µl complete growth medium + 100 ul of the tested compound per well in a 96-well plate for 24 hours before the MTT assay.
Cell culture protocol
1. Culture medium was removed to a centrifuge tube.
2. The cell layer was rinsed briefly with 0.25% (w/v) Trypsin and 0.53 mM EDTA solution to remove all traces of serum which contains Trypsin inhibitor. (Trypsin 0.5 EDTA, 10X15400054)
3. Three ml of Trypsin EDTA solution was added to flask and the cells were observed under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Eight mL of complete growth medium was added and cells are aspirated by gently pipetting.
5. The cell suspension was transferred to the centrifuge tube with the medium and cells obtained from step 1, then centrifuged at approximately 125 xg for 5 to 10 minutes. Then the supernatant was discarded.
6. The cell pellet was resuspended in fresh growth medium and appropriate aliquots of the cell suspension were added to new culture vessels.
7. Cell culture was incubated at 37°C for 48 hours.
Cell counting & cell viability ‘Trypan blue’ by Hemocytometer:
The cells were counted by hemocytometer to estimate the total number of cells.
- Adding 10
of the harvested cells to the hemocytometer
- The chamber was then placed in the inverted microscope under 10X objective.
- The cells in the large, central gridded square (1mm2) were counted, and were multiplied by 104 for the estimation of the total number of cells per ml.
- 0.1 ml of trypan blue solution in buffer were added to 0.1 ml of cells
- The samples were loaded on hemocytometer and examined under low magnification to estimate the number of dead cells.
- The cells were counted and the dead cells were measured to estimate the number of viable cells.
% of viable cell = [1.00 – (number of blue cells Number of total cells)]
100
- The number of viable cells per ml of culture estimated by the formula:
Number of viable cells 104
1.1 = cells/mL culture
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2. Preparation of CAR, siRNA circuits for engineering of T cells
a. The T cell was engineered by two vectors:
· siRNA (BBa_K3244025 ligated to psB1C3)
· CAR-T (BBa_K3244029 ligated to pcDNA 3.1+)
3. Design of the circuit by bioinformatics (Please refer to our pages of Design and Parts)
4. Digestion of plasmids:
a. pcDNA 3.1. using restriction enzymes BglII and EcoRI
b. psB1C3 using restriction enzymes EcoRI and Pst-1
5. Ligation of fragments (Fast Digestion of DNA and ligation protocol from ThermoFisher )
a. Digestion of plasmid
b. Digestion of fragments
c. Ligation of fragments
d. Ligation of construct to plasmid
· Digestion protocol: by fast digestion protocol of DNA there.
· Ligation protocol: Rapid ligation protocol. T4 ligase kit
6. Agarose Gel electrophoresis: for confirmation of products
Agarose Gel Electrophoresis Technique
- Prepare Loading Buffer (TBE): 5X.
- Tris base 5.4 grm.
- Boric acid: 2.75 grm.
- EDTA: 2ml (0.5mol), pH 8.8.
- Dissolve in 100ml distalled water.
- Prepare 100ml 1X Buffer:
- 20ml TBE (5X) added to 80 ml DW.
- Prepare agarose Gel:
- Weight 1 grm agarose.
- Dissolve in 50 ml TBE buffer 1X.
- Heat till boiling in microwave.
- Mix well until agarose dissolved like watery appearance.
- agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis.
4. Pour a gel: After cooling the solution to about 50C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator.
5. Preparation of loading dye: 6X.
- According to manufacture instruction.
- Solidify Gel: the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pippeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually coloured red.
7. Separation of PMNs by Ficoll-Hypaque protocol
Immunofluorescence Protocol
I. Specimen Preparation
1. Cultured Cell Lines:
- Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
2. Allow cells to fix for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining (Section C).
II. Immunostaining
N.B: All subsequent incubations should be carried out at room
temperature unless otherwise noted in a humid light-tight box or covered
dish/plate to prevent drying and fluorochrome fading.
- Block specimen in blocking buffer for 60 min.
- While blocking, prepare primary antibody by diluting as indicated on
in antibody dilution buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
- Rinse three times in 1X PBS for 5 min each.
- 8. Coverslip slides with Prolong Gold Antifade Reagent with DAPI.
- 9. For best results, allow the mounting reagent to cure overnight at room temperature.
- For long-term storage, store slides flat at 4°C protected from light.
III. Microscopic examination
For best results examine specimens immediately or store the slides flat at 4°C protected from light for long term storage. The microscopic examination using appropriate excitation wavelength was performed by LABOMED Fluorescence microscope LX400, cat no: 9126000; USA.
Isolation of mononuclear cells Protocol
Preparation of the sample
Fresh blood should be used to ensure high viability of isolated mononuclear cells. Prepare the sample at 18ºC to 20°C.
1. To a 10 ml centrifuge tube add 2 ml of defibrinated- or anticoagulant-treated blood and an equal volume of
balanced salt solution (final volume 4 ml).
2. Mix the blood and buffer by inverting the tube several times or by drawing the mixture in and out of a pipette.
Procedure for isolation of mononuclear cells
1. Invert the Ficoll-Paque media bottle several times to ensure thorough mixing.
For withdrawal of Ficoll-Paque media by syringe:
Snap-off the polypropylene cap and insert the syringe needle through the septum (Fig 1).
For withdrawal of Ficoll-Paque media by pipette:
Remove the snap-off polypropylene cap. Lift the aluminum ring. Pull off the metal seal. Remove the silver ring, Remove the rubber closure. Using aseptic techniques, withdraw the required volume of Ficoll-Paque media.
2. Add Ficoll-Paque media (3 ml) to the centrifuge tube.
3. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque media solution Important: When layering the sample do not mix the Ficoll-Paque media solution and the diluted blood sample.
4. Centrifuge at 400 g for 30 to 40 min at 18ºC to 20°C (brake should be turned off).
5. Draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the mononuclear cell layer undisturbed at the interface (Fig 4 and Fig 5). The upper layer, which contains the plasma, may be saved for later use.
6. Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette.
Washing the cell isolate
1. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 6 ml) of balanced salt
solution to mononuclear cells in the centrifuge tube.
2. Suspend the cells by gently drawing them in and out of a pipette.
3. Centrifuge at 400 to 500 × g for 10 to 15 min at 18°C to 20°C.
Note: centrifugation at high speed increases the mononuclear cell recovery. However, if it is important to also get rid
of platelets a lower centrifugation speed is recommended (60 to 100 × g).
4. Remove the supernatant.
5. Resuspend the mononuclear cells in 6 to 8 ml balanced salt solution.
6. Centrifuge at 400 to 500 × g (or 60 to 100 × g for removal of platelets) for 10 min at 18°C to 20°C.
7. Remove the supernatant.
8. Resuspend the cell pellet in media appropriate for the application.
8. Isolation of CD3/CD28 positive T cell population using Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation# (2 mL), CAT No. 11131D.
9. Knockout of PD-1 in isolated T-cells by lipofectamine 3000 followed by immunofluorescence and flow cytometry using PD-1 specific antibody
10. Culture of knockout T-cells by optimizer T cell expansion media
(OpTmizer™ T Cell Expansion SFM with 100 U/mL penicillin/streptomycin CAT NO. A104850)
11. T-cell engineering for CAR-T using CRISPR editing protocol for T cells
12. Co-culture of engineered T cell with bladder cancer cell line
13. Cell count/cell viability by Trypan blue
14. Cell cytotoxicity assay The cell cytotoxicity assay was performed using the Vybrant MTT Cell Proliferation Assay Kit (Thermo Scientific, USA)
Cell Cytotoxicity assay:
The cell cytotoxicity assay was performed using the Vybrant MTT Cell Proliferation Assay Kit, (Thermo Scientific, USA)
8. Vybrant MTT Cell Proliferation Assay (MTT 3-4-5 Dimethylthiaz 1gm CAT. No. M6494)
A. Reagent Preparation
- Twelve mM of MTT stock solution was prepared by adding 1 mL of sterile PBS to one 5 mg vial of MTT (Component A), Mixed by vortexing until dissolved. Once prepared, the MTT solution can be stored for four weeks at 4°C protected from light.
- Ten mL of 0.01 M HCl was added to one tube containing 1 gm of SDS (Component B), the solution gently mixed by inversion or sonication until the SDS dissolves. Once prepared, the solution should be used promptly. Each tube makes sufficient solution for 100 tests, using 100 µL per well.
- Culturing Cells
The culture conditions used to grow the cells can affect the results and must be taken into consideration when analyzing the data. The age of the cultures, number of passages and details of the growth medium can all be important factors. Natural variation in the requirements and growth rates of different cell lines make it difficult to provide precise guidelines for preparing your cells. In general, cells seeded at densities between 5000-10,000 cells per well should reach optimal population densities within 48-72 hours. Note that the presence of phenol red in the final assay samples can seriously affect results. We strongly recommend that the cells be cultured in medium free of phenol red, if possible. Alternatively, the final incubation with the MTT can be performed after exchanging the cells into medium free of phenol red.
C. Labeling Cells
- For adherent cells, the medium was removed and replaced with 100 µL of fresh culture medium. For non-adherent cells, the microplate was centrifuged, pellet the cells, carefully remove as much medium as possible and replace it with 100 µL of fresh medium.
- Add 10 µL of the 12 mM MTT stock solution (prepared in step 1.1) to each well. Include a negative control of 10 µL of the MTT stock solution added to 100 µL of medium alone.
- Incubate at 37°C for 4 hours. At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.
- Hundred µL of the SDS-HCl solution (prepared in step 1.2) was mixed thoroughly to each well using the pipette.
- Incubate the microplate at 37°C for 4 C hours in a humidified chamber. Longer incubations will decrease the sensitivity of the assay.
- Each sample was mixed again using a pipette and read absorbance at 570 nm.
15. Detection of cell apoptosis using Annexin V staining kit by flow cytometry
Annexin V staining protocol by Flow cytometry
Experimental Procedure
1. 10X Binding Buffer was diluted to 1X using distilled water (1 mL 10X Binding Buffer + 9 mL dH20).
2. Cells was washed once in PBS, then once in 1X Binding Buffer.
3. Cells was resuspended in 1X Binding Buffer at 1-5x106/mL.
4. Five 5 µL of fluorochrome-conjugated Annexin V was added to 100 µL of the cell suspension. (ANNEXIN V APOP DETECT Kit APC 50 test, CAT No. 88-8007-72)
5. Cells were incubated for 10-15 minutes at room temperature.
6. Cells were washed in 1X Binding Buffer and resuspend in 200 µL of 1X Binding Buffer.
7. 5 µL of Propidium Iodide Staining Solution (cat. 00-6990) was added
8. Analyze by flow cytometry within 4 hours, storing at 2-8°C in the dark
16. Detection of PD-1 silencing in engineered T cells by immunofluorescence