T cells; Apoptosis

- At a concentration of 10 µg/mL, PHA-P stimulated positively selected decidual CD8+ T lymphocytes (1×10⁶ cells/mL) to produce cytokines. After 24 and 48 hours of incubation, detectable levels of CSF2, IFNG, IL1B, IL2, IL6, IL8, IL10, IL12, and TNF were found in the culture supernatants, while IL4 levels were below the detection range. Specifically, PHA-P-stimulated decidual CD8+ T lymphocytes produced high levels of IFNG and IL8, and low levels of GM-CSF, IL-1β, IL-2, IL-6, IL-10, IL-12, and TNF. [1]
- In a placental explant invasion assay, PHA-P (10 µg/mL) was used as a control. When placental explants were cultured with PHA-P-containing medium in the upper chamber for 7 days, there was no difference in the number of invaded extravillous trophoblast cells compared to medium-alone controls. [1]

---

Phytohemagglutinin-P (10 μg/ml; 24–48 hours)-stimulated decidual CD8+ T cells produce modest quantities of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor, and high levels of both interferon gamma and interleukin (IL) 8[2].

---

In vitro, PHA-P demonstrates potent T lymphocyte mitogenic activity. At concentrations ranging from 5 to 10 μg/mL, it effectively stimulates the proliferation of human peripheral blood mononuclear cells (PBMCs) and T cells. PHA-P (10 μg/mL; 24–48 hours) induces the production of various cytokines from stimulated decidual CD8+ T cells, including modest quantities of GM-CSF, IL-1β, IL-2, IL-6, IL-10, IL-12, TNF-α, and notably high levels of IFN-γ and IL-8. The mitogenic potency threshold for human PBMC stimulation is ≤10 μg/mL as measured by bromodeoxyuridine incorporation.

Male Sprague-Dawley rats given Phytohemagglutinin-P (ip; 25 and 50 mg/kg; daily for 15 days) exhibit a reduction in the growth of longitudinal bones, chondrocyte counts, cartilage plate thickness, metaphyseal mass of hard tissue, percentage of calcified cartilage core, and number of osteoblasts per millimeter of bone surface. Furthermore, PHA-P raises the average number of nuclei per osteoclast, the number of labeled osteoclastic nuclei, and the total number of osteoclasts[ 1].

---

In vivo, PHA-P exhibits both immunostimulatory and context-dependent immunosuppressive effects. In mice, intraperitoneal administration effectively suppresses skin allograft rejection across the H-2 locus, increasing graft survival by 70–130% without significant mortality or apparent toxicity. Conversely, in rat models, intraperitoneal injection of PHA-P (25 and 50 mg/kg; daily for 15 days) reduces longitudinal bone growth, chondrocyte counts, and cartilage plate thickness while increasing osteoclast numbers. In Trichinella spiralis-infected mice, intravenous PHA-P (10 mg/kg) administered 24 hours prior to infection stimulates apoptosis in jejunal mucosa and muscular inflammatory infiltration, resulting in reduced muscle larvae counts compared to infected controls.

As a lectin, PHA-P functions through carbohydrate-binding rather than classical enzyme/receptor interactions. Standard hemagglutination assays are employed to characterize its binding activity: serial two-fold dilutions of PHA-P in phosphate-buffered saline (PBS) are prepared in V-bottom 96-well plates. An equal volume of a 2% suspension of washed human or animal erythrocytes (typically type O human red blood cells) is added to each well. The plate is gently mixed and incubated at room temperature for 1–2 hours. Hemagglutination is assessed visually as a diffuse mat of agglutinated cells, with the titer defined as the highest dilution showing positive agglutination. Inhibition assays using specific monosaccharides (e.g., D-mannose, N-acetyl-D-galactosamine) can determine carbohydrate-binding specificity.

- Cytokine Production Assay: Positively selected CD8+ decidual lymphocytes were resuspended in complete medium at a concentration of 1×10⁶ cells/mL. 100 µL of this cell suspension was dispensed into wells of round-bottomed 96-well plates. An equal volume of PHA-P at a concentration of 10 µg/mL was added to stimulate the CD8+ decidual lymphocytes. The plates were then incubated for 24 and 48 hours. Following incubation, supernatants were collected, centrifuged at maximum speed in a microcentrifuge for 2 minutes to obtain cell-free supernatants, which were stored at -70°C for subsequent cytokine analysis. [1]
- Invasion Assay (Control): Placental explants were established on Matrigel-coated 8-µm pore filters. 200 µL of complete medium was added to the lower chamber. On the following day (Day 1), 200 µL of PHA-P (10 µg/mL) containing DMEM-F12 medium was dispensed into the upper chamber of control wells, ensuring villous explants were covered. Plates were then cultured at 37°C in a 5% CO₂ incubator for 7 days. After 7 days, the Matrigel and explants were removed, and the upper side of the membrane was cleaned. Filters were fixed in 10% neutral buffered formalin, stained with hematoxylin and eosin, and mounted. The total number of cells on the underside of the filter was counted at x100 magnification for the entire area. There was no difference in the number of invaded extravillous trophoblast cells between the PHA-P control and medium-alone controls. [1]

---

A typical protocol for PHA-P-stimulated PBMC proliferation uses human peripheral blood mononuclear cells isolated by Ficoll density gradient centrifugation. Cells are seeded at 2 × 10⁶ cells/mL in complete culture medium (DMEM or RPMI-1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and Glutamax) in 24-well plates. PHA-P is added at a final concentration of 5–10 μg/mL. Cultures are incubated at 37°C in 5% CO₂ for 24–72 hours depending on the endpoint. For proliferation assessment, 5-bromo-2′-deoxyuridine (BrdU) incorporation assay can be performed after 48–72 hours, or cell viability can be measured using MTT or flow cytometry. For cytokine analysis, culture supernatants are collected at 24–48 hours for ELISA or multiplex assays.

A representative in vivo study of PHA-P employs a murine allograft rejection model. Female mice (e.g., C3H and C57BL/6 strains) receive a large intraperitoneal (i.p.) priming dose of PHA-P (dose ranges reported from 10–50 mg/kg depending on study) prior to skin grafting, followed by smaller daily i.p. injections post-grafting. Graft survival is monitored daily by visual inspection, with rejection defined as complete graft necrosis. For immunomodulation studies in infection models, PHA-P (10 mg/kg) is administered intravenously 24 hours prior to pathogen challenge. Endpoints include lymphocyte apoptosis analysis by flow cytometry (Annexin-V staining), tissue histology (TUNEL assay), and pathogen load quantification.

Specific pharmacokinetic data for PHA-P are limited in standard literature. As a high molecular weight glycoprotein (approximately 110–150 kDa), PHA-P is not expected to be significantly absorbed through the oral route. Following parenteral administration (intravenous or intraperitoneal), it likely distributes primarily within the vascular and lymphoid compartments due to its size and lectin properties. Metabolism is presumed to occur via proteolytic degradation, though detailed parameters such as half-life (t₁/₂), volume of distribution (Vd), and clearance (CL) have not been systematically characterized in published pharmacokinetic studies.

PHA-P exhibits a moderate toxicity profile. Acute toxicity is primarily associated with its hemagglutinating activity, which can lead to erythrocyte clumping and microvascular complications at high doses. In animal studies, intraperitoneal administration at doses up to 50 mg/kg in rats produced no reported mortality but did cause significant effects on bone morphology. Notably, PHA-P has been described as having "no apparent toxicity" in murine allograft studies at immunosuppressive doses. In humans, clinical use (10–40 mg/day) may cause transient allergic reactions, with rare cases of anaphylactic shock reported. Oral ingestion of undercooked red kidney beans containing PHA can induce food poisoning characterized by nausea, vomiting, and diarrhea.

[1]. Effector activity of decidual CD8+ T lymphocytes in early human pregnancy. Biol Reprod. 2006;75(4):562-567.

- PHA-P at 10 µg/mL was used as a stimulant to induce cytokine production from decidual CD8+ T lymphocytes and as a control in trophoblast invasion assays. [1]
- The supernatants from PHA-P-stimulated decidual CD8+ T lymphocytes (48-hour conditioned medium) were used to assess the effect on trophoblast invasion. In contrast to the PHA-P control alone, these supernatants significantly increased the capacity of extravillous trophoblast cells to invade through Matrigel (mean number of invaded cells normalized to PHA-P control: 280 ± 61.3 vs. 100 ± 0, P = 0.006). [1]

9008-97-3

Phytohemagglutinin;9008-97-3

596533

Typically exists as White to light yellow solids at room temperature

1

6

5

25

419

0

PHA-P

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~10 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)