Phytohemagglutinin-P (PHA-P), sterile lyophilized
Phytohemagglutinin-P (PHA-P) is a sterile lyophilized protein purified from selected seed beans of Phaseolus vulgaris. PHA may be used to stimulate mitotic division of lymphocytes, in cytogenetic and immunological studies.
It is a fine crystalline powder in the white tablet form. At dissolved in purified water, it forms a colorless transparent or slightly cloudy solution, pH from 7.0 to 7.4. It contains lactose as a stabilizer.
This product is intended for research purposes. The product has a Russian registration certificate as a medical product.
Preparation instructions:
PHA may be used to stimulate mitotic division of lymphocytes. Each bottle of lyophilized PHA-P should be reconstituted by adding 0.9% sterile NaCl or sterile purified water to final concentration PHA-P 1 mg/ml.
To achieve the PHA-P optimal concentration for cytogenetic studies, 0.05-0.1 ml of PHA-P solution with a concentration of 1 mg /ml is added into the medium for whole blood cultivation (for obtain final concentration 6-12 mkg/ml of PHA-P).
Whole blood or separated leukocytes have been used for cytogenetic studies, but the former is the simplest and most widely used in routine studies. Serum should be screened prior to use to ensure the selection of batches free of inhibitory effects.
1. Using aseptic technique, add to a sterile culture vial:
- 6.0 ml of cultural medium (DMEM Cat No C410п, RPMI-1640 Cat No C310п)
- 1.5 ml of serum (bovine serum or fetal bovine serum)
- 100 µL of PHA-P solution
- 0.5 ml of heparinized blood
2. Gently mix and incubate the culture at 37°C for 72 hours. Mix each bottle by inversion daily.
3. Remove culture bottles from the incubator 1.5 hours prior to harvesting. Add 50µL of a Colchicine solution (100 µg/mL) to each culture (Colchicine 0.5 mg, Paneco-ltd Cat No Ф071 dissolved in 5 ml sterile water). Mix gently and return to the 37°C incubator.
4. 1,5-2 hours after adding Colchicine, remove the cultures from the incubator and transfer to plastic graduated centrifuge tubes marked with sample details.
5. Centrifuge the cultures in a bench centrifuge at 500x-1000x g for 5-7 minutes. Remove most of the supernatant fluid and discard.
6. Resuspend the pellet in 10 mL of 75 mM potassium chloride solution pre-warmed to 37°C for 10 minutes.
7. Centrifuge as in step 5 and discard supernatant (leave ~ 0.5-1.0 ml of supernatant).
8. Using a Pasteur pipette slowly add 6 to 8 mL of freshly prepared acetic alcohol (3 volume of methyl alcohol: 1 volume of acetic acid) to the pellet while agitating constantly on a vortex mixer. Add the fixative dropwise at first, followed by a slow trickle to minimize cellular damage and formation of lumps. Leave at 4°C for 10 minutes.
9. Centrifuge lightly, remove the supernatant fluid as before and slowly add an additional 5 mL of cold acetic alcohol to resuspend pellet.
10. Repeat step 9 twice more, re-suspending finally in 0.5 -1.0 mL acetic alcohol. Use this cell suspension to prepare slides for examination. Care must be taken to avoid agitation of the cells.
11. Apply 1 or 2 drops of the resuspended cell preparation to the center of a prepared glass slide and allow to spread.
12. Stain with Giemsa or 2% acetic acid-orcein stain, etc. according to the standard protocol.
HOW SUPPLIED: in sterile penicillin vials by 1 mg, 5 mg or 10 mg.
SHELF LIFE: 1.5 years.
STORAGE: +2 to +8 °C.
RECOMMENDED USE:
- Do not use the product after its expiry date.
- Use gloves, mask and clothes adapted to the manipulation of the product to avoid contamination.
- Manipulate the product in aseptic conditions.
